Thyroid cancer (TC) is the most common secondary tumor in patients diagnosed with primary breast cancer (BC) [1
]. Similarly, BC is reported as the most frequent second primary tumor in TC patients [3
]. The lifetime risk for these cancers is increased in patients with a positive family history of both TC and BC [3
], and clinical cancer surveillance might be appropriate for some of the cases [8
]. In a large cohort of 13,798 BC Chinese patients, family history of cancer was the only predictor of secondary TC in BC patients [1
]. Studies to investigate mechanisms involved in this association are necessary [9
Hereditary BC and TC are mainly related to Cowden syndrome in which 30% to 35% of patients are positive for PTEN
pathogenic variants [10
]. Breast cancer is the most frequent cancer in the Li–Fraumeni syndrome tumor spectrum, which is associated with pathogenic variants in TP53
]. However, TC is also rarely described in Li–Fraumeni patients [13
]. In addition to PTEN
, other candidates were reported as presenting potential predisposition genes associated with familial BC/TC [14
]. In Cowden or Cowden-like syndromes, SDHx
genes were reported as modifiers of the phenotype [16
]. Two variants mapped in PARP4
(p.G496V and p.T1170I) were detected among 14 unrelated individuals diagnosed with both BC and TC [14
]. Four Polish founder variants in CHEK2
(1100delC, IVS2+1G>A, del5395, and I157T) were described in TC patients who were also diagnosed with BC or had familial breast cancer history [15
An association between BC and TC was also described in TC patients treated with surgery and exposed to radioiodine therapy. These patients presented a higher risk of developing a second primary cancer of the breast [18
]. A plausible explanation is a deregulation of thyroid hormones (in TC and in other thyroid dysfunctions such as hyperthyroidism and hypothyroidism), which may have pro- and anti-oncogenic properties able to trigger BC development [19
]. A recent study based on United States survivors (2000–2015) showed an increased risk of second primary papillary TC for several cancer types, including BC. According to these authors, the risk of developing papillary TC was not clearly associated with the treatment of the first tumor and shared risk factors could explain this association [20
]. High-penetrance genes or genetic variants associated with this phenotype are poorly explored, and markers for preventive screening would benefit high-risk patients. Herein, the germline DNA of patients diagnosed with BC and/or TC and familial history of these tumors was whole-exome sequenced to investigate genetic variants potentially associated with hereditary BC and TC.
An association between TC and BC as primary tumors, treated with surgery only, and second primary tumors in young patients, below 40-years-old, was reported as early as 1984, in which a common etiological factor was suggested [31
]. A population-based study reported a significantly increased risk of BC and TC for relatives of patients with these tumors [7
]. Further studies demonstrated an increased association for the co-occurrence of BC and TC in the same patient, likely due to treatment for the primary site, and in patients having a positive family history of BC and TC [3
]. Current genetic evidence for this association is restricted to Cowden syndrome and, in a small proportion, to Li–Fraumeni syndrome, involving pathogenic variants in PTEN
, respectively [13
]. However, these variants only explain a small proportion of familial cases. Our findings provide evidence that the MUS81
c.1292G>A (p.R431H) may explain, at least in part, the familial BC and TC clustering.
We identified variants in cancer-related genes [21
] that could potentially play a role in familial BC and TC, as well as MUS81
c.1292G>A penetrance moderator in three patients (M1, M3, and M4) that had both the MUS81
c.1292G>A and an additional variant in a cancer-related gene. Although patients M3 and M4 had no personal history of polyposis, both patients had the MUTYH
c.1187G>A (p.G396D) variant. This variant is described as partially defective [33
] and is one of the most common variants, together with p.Y179C, associated with bi-allelic MUTYH
mutation and the recessive form of familial polyposis [34
]. Of note, the father of patient M4 had colorectal cancer. Patient M4 also had a variant in ERRC3
(c.2077C>T; p.L693F) that was only reported in the single-nucleotide polymorphism database (dbSNP).
Patient M1 had three variants in cancer-related genes (PRKAR1A
c.7313C>G; and MTAP
c.634T>C). Inactivating mutations of PRKAR1A
in TC and, in less frequency, in pancreatic adenocarcinoma patients are associated with Carney Complex syndrome [36
]. The PRKAR1A
variant identified here is described as likely benign in ClinVar because of its higher than expected populational frequency; however, functional assays are necessary to validate this classification. Patient M1 had a family history (maternal and paternal) of pancreatic carcinoma. Variants in COL7A1
were not reported in ClinVar; however, variants in these genes are related to squamous cell carcinoma (as part of epidermolysis bullosa spectrum) [37
] and sarcoma [38
Our network analysis revealed that, among 17 cancer-related genes, MUTYH and PRKAR1A indirectly interact with MUS81, via MSH2 or CHECK2, while ERRC3 directly interacts with MUS81. The top significant pathways with related to cancer and MUS81 were DNA replication, nucleotide excision repair, and lagging strand synthesis. These altered genes can act synergistically and potentially dysregulate the same pathways.
Other recurrently altered genes were detected but, to our knowledge, they were involved in pathways not directly linked to cancer. For instance, the kinesin protein KIF19
is associated with the transport of membranous organelles and protein complexes in a microtubule- and ATP-dependent manner [39
Recently, three Brazilian families with hereditary papillary TC investigated by whole-exome sequencing showed pathogenic/likely pathogenic variants in ANXA3
, and SAPCD1
]. We found variants of unknown significance in ANXA3
in two patients negative for MUS81
c.1292G>A variant. The authors also reported one family with BC history that harbored SERPINA1
c.1087G>T (p.G172W) variant. Interestingly, here, we found a pathogenic SERPINA1
variant (c.1096G>A; p.E366K) in patient W14. SERPINA1
is a potential candidate associated with the risk of developing familial BC and TC.
Breast and thyroid cancer tissues from patients with germline MUS81
c.1292G>A (p.R431H) exhibited negative or lower MUS81 expression levels compared with wild-type BC, and normal breast and thyroid tissues. This suggests that MUS81 activity is compromised in tumors of patients positive for p.R431H variant, and conservation of the amino acid is essential for protein function and stability. The MUS81 protein acts in 3′-flap stalled replication fork [41
], Holliday structure [42
], which prevents chromosomal breaks and deleterious recombination [43
]. Deregulated expression of MUS81
was reported in serous ovarian [45
] and prostate [46
] cancer cells. In hepatocellular [47
] and BC cells [48
], depletion of MUS81 increased chemosensitivity, highlighting a potential target for cancer treatment. Due to its function in DNA repair, MUS81
is considered one of the guardians of genome integrity [25
Consistent with our results in MUS81 protein expression, our functional assays revealed that MUS81 p.R431H is significantly more rapidly degraded than its wild-type counterpart in thyroid and glioblastoma cell models. MUS81 p.R431H also affected the DNA damage response, and its overexpression in cell models was associated with an increase in DNA damage. The analysis of all exons of MUS81
in the tumor sample from patient M1 confirmed the presence of both germline variants c.1292G>A and c.344C>T. No somatic point mutation was identified. Nonetheless, c.1292G>A might explain the low or negative expression of MUS81 in the tumor, as shown in immunohistochemistry analysis. Of note, MUS81
was described as a tumor suppressor with a haploinsufficient phenotype [49
]. Other mechanisms, such as copy number alteration or DNA methylation, could be involved in the MUS81
regulation. Published data from BC and TC [51
] showed MUS81
deletion events and their association with gene down-expression. Moreover, high DNA methylation levels in sites outside the MUS81
CpG island could also regulate gene expression (Figure S6
). Further studies are necessary to address mechanisms involved specifically with the MUS81
c.1292G>A p.R431H regulation.
c.1292G>A (p.R431H) being reported with MAF = 0.018 in public databases, a significantly higher frequency was detected in our familial group of patients, compared with the healthy Brazilian population (p
= 8.7 × 10−4
) and with sporadic BC and TC tissues (p
= 6.3 × 10−3
). Recently, a consensual and more stringent MAF threshold was used to facilitate the identification of new pathogenic variants that predispose to rare cancer syndromes. Causal variants classified as pathogenic by the HGMD (Human Gene Mutation Database) and ClinVar databases often have MAF < 0.01% [53
]. Nevertheless, a thorough investigation of potential new mutations and/or genes that may display variable penetrance is a valid approach, given that risk alleles may still be hidden in population databases used as controls [54
]. For instance, a potentially pathogenic mutation of TP53
was highly prevalent in population databases, with higher frequencies than previously expected [55
]. This finding pointed out the existence of penetrance modifiers even in genes broadly studied in cancer, such as TP53
. We suggest that MUS81
c.1292G>A (p.R431H) plays a role in familial breast and thyroid cancer risk. Replication of these findings in larger sample sets is needed to elucidate the penetrance of this variant.
Another important note is that TC is less incident in males compared to females [56
], and clinical manifestation of hereditary cancer syndromes is often related to cancer diagnosis in the less affected sex, compared to sporadic cancer cases [58
]. The brother of the index patient M5 was diagnosed with thyroid cancer at 36 years old, which is considered early onset for sporadic thyroid cancer [56
This study has several limitations. We were unable to confirm the cancer diagnosis in carriers of the MUS81 variant in relatives of the patient M2 (M2-3 and M2-2) since reports of prophylactic thyroidectomy were provided by the index patient. Although the stringent inclusion criteria strengthened the specificity of our findings, the number of families investigated is small. Furthermore, selecting the most recurrent variant might have introduced bias when comparing the allele frequency between the familial cohort and healthy Brazilian individuals. Other studies using a large cohort of patients and families with a similar phenotype are necessary to validate our findings. We found pathogenic/likely pathogenic variants in cancer-related genes that could contribute to the BC and TC phenotype (e.g., MUTYH c.1187G>A and SERPINA1 c.1096G>A). Unfortunately, the affected relatives of our index patients were treated in other institutions and were not accessible for genetic testing. Future studies would help to clarify whether these variants are associated with BC and TC and/or interact with MUS81, contributing to the cancer phenotype. Moreover, investigation of MUS81 protein expression is an important next step to validate the effect of MUS81 c.1292G>A in a wide cohort of familial and sporadic BC and TC samples.
4. Materials and Methods
In a preliminary survey, we evaluated the clinical data and family history of 130 patients with personal and family history of BC and TC. Then, we established the following patient inclusion criteria: index case with non-medullary TC and/or BC with family cancer history of these cancers AND at least two first- or second-degree relatives with one of these cancers developed before the age of 45 OR the index case plus one first-degree relative affected with one of these cancers with age of cancer onset lower than 40 years old. Exclusion criteria comprised female patients diagnosed with TC and a previous history of BC treated by radiotherapy. Medullary thyroid cancer was excluded due to its association with familial medullary thyroid carcinoma syndrome associated with RET mutations (OMIM155240).
Based on these criteria, we selected 20 unrelated patients with a personal and family history of TC and/or BC, which were followed prospectively at the Department of Oncogenetics of the A.C. Camargo Cancer Center, Sao Paulo, Brazil. Subjects provided written informed consent following the Declaration of Helsinki and were advised of the procedures. The study was approved by the institutional Human Research Ethics Committee (FMB-PC-197/2012; CEP1175/08ext).
The medical records reported results of genetic testing for three patients as follows: one patient was tested for TP53
p.R337H (Brazilian founder mutation for Li–Fraumeni syndrome [59
]) and was negative for this variant; one patient tested negative for TP53
whole-gene, but there was no technical detail; one patient with a putative diagnosis of Cowden syndrome was negative for PTEN
variants with no further details.
Patients were identified with an alphanumeric character being either F (family with more than one patient assessed by exome sequencing), M (patients positive for MUS81
c.1292G>A p.R431H), or W (patients MUS81
wild-type), followed by Hindu-Arabic numerals (1–20). Clinical features and cancer family history of the index cases are detailed in Table S1
4.2. DNA Isolation and Library Construction
Genomic DNA from blood samples (index patients and healthy individuals), saliva (recruited relatives), and frozen tumor tissues (sporadic BC and TC) were extracted using a Qiacube DNA Blood kit (Qiagen, Valencia, CA, USA), Oragene-DNA kit (DNA Genotek, Ottawa, ON, Canada), and Gentra Puregene Tissue Kit (Qiagen, Valencia, CA, USA), respectively. Six microdissected FFPE tissue samples were submitted to deparaffinization, protease digestion, and total DNA extraction using a RecoverAll™ Total Nucleic Acid kit (Ambion, Thermo Fisher Scientific, Waltham, MA, USA). DNA library preparation and whole-exome sequencing were carried out using the Exome Nextera Enrichment kit (Illumina Inc, San Diego, CA, USA) according to the manufacturer’s recommendations and sequenced on Illumina HiSeq 2000 (Illumina Inc, San Diego, CA, USA).
4.3. Whole-Exome Sequencing, Bioinformatics Analyses, and Variant Prioritization
Paired-end (PE) raw sequencing data were constructed and sequenced using the Illumina HiSeq 2000 platform (Illumina Inc, San Diego, CA, USA). Read length was 100 bp. PE raw sequencing data had the adaptors trimmed using TrimGalore with default parameters in paired-end mode (https://www.bioinformatics.babraham.ac.uk/
), and reads were aligned to the GRCh37/hg19 human reference assembly using BWA-mem version 0.7.15 with default parameters [60
]. Duplicate reads (multiple reads that start and end at the same position potentially due to amplification artefacts) were flagged using SAMBAMBA markdup (https://lomereiter.github.io/sambamba/
), and base quality scores of the aligned reads were recalibrated using GATK v3.6-0 [61
]. Alignment statistics were obtained with Picard (http://picard.sourceforge.net/
), SAMtools samtools [62
], GATK, and BAMtools (https://github.com/pezmaster31/bamtools
). Variants were called with GATK HaplotypeCaller in gVCF mode and genotypes were called for the entire cohort using GATK genotypeGVCFs. Variants were annotated using ANNOVAR [63
] and Varseq v2.x (Golden Helix, Inc., Bozeman, MT, USA, www.goldenhelix.com
We excluded variants as follows: (1) synonymous; (2) observed in more than 2% of the analyzed alleles in GnomAD or AbraOM [64
]; (3) with allele fraction <30% (fraction of reads supporting the variant); (4) variants with <10 reads for SNPs and <10 reads for INDELs; (5) variants not shared by all individuals of the same family. This last criterion (5) was adopted for variants detected in the families F1 (W6, W6-1, and W6-2) and F2 (W7 and W7-1).
In silico prediction tools were used to classify the variants and their pathogenicity score. For missense and loss-of-function variants to be predicted as pathogenic in silico, we used CADD with the threshold > 20 [65
], while for splice site we used Ada and RF, with the threshold > 0.6 [66
]. We also excluded variants classified as benign in ClinVar. We then flagged genes ranked according to high mutation burden due to extensive gene length [24
]. From a total of 3855 remaining variants, we focused on variants mapped in cancer-related genes [18
], recurrently altered genes, and recurrent variants detected in more than one patient/family. A summary of variant prioritization is described in Figure S1
. The relationship between the 17 cancer-related genes and MUS81 was illustrated in a PPI network using data from the IID v. 2018-11 [27
). We retrieved all direct physical interactions (experimentally identified in human, orthologs from other organisms, and computationally predicted). Final network was visualization using NAViGaTOR version 3.13 [67
]. We also used the 17 cancer-related genes and MUS81
, to perform a pathway enrichment analysis using PathDIP version 188.8.131.52 (Database version 184.108.40.206) [28
]. An adjusted p
-value was obtained using Bonferroni correction at a significance level <0.05.
4.4. Data Confirmation
Sanger sequencing analyses were performed to (1) confirm the MUS81
c.1292G>A variant in five index patients (M1, M2, M3, M4 and M5) and four relatives (M2-1, M2-2, M2-3, and M5-1), (2) confirm the MUS81
c.1168A>C variant detected in one patient (case M4), and (3) investigate all MUS81
exons from the tumor of patient M1, which was the only sample available from fresh frozen tissue. Forward and reverse primer sequences are listed in Table S6
. In summary, after amplification by conventional PCR, sequencing was performed using the Applied BigDye®
Terminator v3.1 Cycle Sequencing Kit protocol (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). The Prism 3130XL sequencing apparatus (v3.1 Cycle Sequencing, Applied Biosystem, Foster City, CA, USA) was used to run the experiments according to standard protocols. Electropherograms were visualized in CLC Main Workbench (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA).
DNA target genotyping (TaqMan®
SNP assay, ID:C_90491711_10, Thermo Fisher Scientific, Waltham, MA, USA) of MUS81
c.1292G>A was assayed in peripheral blood samples from healthy individuals (N
= 362) and DNA from fresh frozen sporadic BC (N
= 41) and TC (N
= 47) (Table S5
). Statistical analyses were performed using Fisher’s exact test in R version 3.4.3 R. We also confirmed the MUS81
c.1292G>A status in FFPE samples used in the immunoexpression assays (described below), including normal and tumor samples, as well as the tumors of patients M1, M4, and M5. DNA was amplified in the 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with the following cycle conditions: 50 cycles of 95 °C for 10 min, 92 °C for 15 s, and 60 °C for 2 min. The allelic discrimination analysis was performed by Genotyping App in the Thermo Fisher Cloud platform (Applid Biosystems, Foster City, CA, USA).
4.5. Immunohistochemistry Analysis (IHC)
Six FFPE tissues were assessed for MUS81 protein immunoexpression including three MUS81 wild-type tissues (one normal thyroid, one normal breast, and one BC) and three MUS81 p.R431H tumor tissues (one BC from patient M4, one BC from patient M5, and one TC from patient M1). Tissue sections (4 mm thick) were dewaxed and hydrated, followed by antigen retrieval using Tris-EDTA (tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid) pH 9.0 solution in a pressure cooker (Pascal®
, Dakocytomation, Agilent Technologies Inc, Santa Clara, CA, USA). The slides were incubated with methanol containing 0.3% H2
to block the endogenous peroxidase activity. Subsequently, slides were cooled down, and sections were incubated with protein block solution (Protein Block®
Dakocytomation, Agilent Technologies Inc, Santa Clara, CA, USA) for 30 min. Sections were incubated with anti-MUS81 monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA) (Table S7
) (1:50 dilution) at 4 °C overnight. After rinsing with PBS (phosphate buffered saline), the HRP (horseradish peroxidase)-conjugated secondary antibody was added (Envision, Dakocytomation, Agilent Technologies Inc, Santa Clara, CA, USA) for 1 h at room temperature. All sections were visualized using 3-3′-diaminobenzidine (DAB, Dako Cytomation, Carpinteria, CA, USA) and counterstained with Harris hematoxylin (Millipore, Burlington, MA, USA). The negative control consisted of replacing the primary antibody by mouse immunoglobulin, followed by identical staining procedures. MUS81 expression was classified in scores (0–4) according to the percentage of cells positivity: score 0, negative expression; score 1, <10%; score 2, 11% to 25%; score 3, 26% to 50%; score 4, ≥51%. The intensity of the protein expression was considered as weak (score 1), moderate (score 2), or strong (score 3). The final staining score was calculated adding both scores (range 2–7).
4.6. Functional Assessment of MUS81 c.1292G>A
Human thyroid carcinoma (TPC1) cells (BRAF wild type) were obtained from Janete Maria Cerutti (Federal University of São Paulo, Brazil) and subjected to STR analysis [68
]. The human glioblastoma carcinoma (U87-MG) cells were purchased from the American Type Culture Collection (ATCC, lot number 5018014). Both cell lines were kept at very low passages and monthly tested to ensure the absence of Mycoplasma
. TPC1 and U87-MG cell lines were cultured in RPMI (Roswell Park Memorial Institute) 1640 medium (Gibco, Gaithersburg, MD, USA) and Dulbecco’s modified Eagle’s medium with low glucose (Gibco, Gaithersburg, MD, USA), respectively. The medium was supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 250 ng/mL amphotericin B at 37 °C in a humidified 5% CO2
To obtain the MUS81 c.1292G>A sequence, a pCMV6-Entry vector containing a MUS81 (NM_025128) Human cDNA ORF (RC203373, OriGene Technologies, Inc, Rockville, MD, USA) was used in site-directed mutagenesis. The c.1292G>A variant was introduced into the wild-type MUS81 cDNA using sense (5′–CACCCTACGCAGCCACCCCTGGGGAACC–3′) and antisense (5′–GGTTCCCCAGGGGTGGCTGCGTAGGGTG–3′) mutated primers (mutated nucleotides are indicated in bold). The reactions were performed using Pfu DNA polymerase (Stratagene, La Jolla, CA, USA) with the following conditions: 95 °C for 1 min, 25 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 3 min, and final extension of 72 °C for 5 min. The vector containing MUS81 double-stranded mutant and wild-type DNA was transformed into JM109 Competent Cells (Promega, Fitchburg, WI, USA). Next, the DNA was extracted and purified (NucleoBond® Xtra Midi/Maxi; Macherey Nagel, Bethlehem, PA, USA) using the Wizard Plus SV Miniprep DNA Purification System (Promega, Fitchburg, WI, USA). Successful incorporation of the c.1292G>A variant was confirmed by Sanger sequencing using the BigDye® Terminator v3.1 Cycle Sequencing (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) and The Prism 3130XL sequencer (Applied Biosystem, Foster City, CA, USA).
The cell lines, U87-MG (8 × 104 cells) and TPC1 (5 × 104 cells) were cultured in plates with 9 cm2 surface area. After 24 h, cells were transfected with the constructs (2.5 μg into U87-MG and 3 μg into TPC1) containing the wild-type sequence (here designed as MUS81), the mutant sequence (MUS81 c.1292G>A; p.R431H), and empty vector using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s recommendations. At 24 h post-transfection, cells were incubated for one, three, and six hours with 100 μM cycloheximide (CHX) diluted in DMSO (dimethyl sulfoxide) (Merck & Co, Kenilworth, NJ, USA). The same volume of DMSO was used as a negative control, collected 1 h after incubation. At the time points, cells were taken and processed, and the MUS81 protein was detected by immunoblotting. The experiments were performed in triplicate.
After the drug treatment, U87-MG and TPC1 cells were washed with cold PBS (pH 7.4), and cell extracts were prepared in ice-cold lysis buffer (50 mM Tris, pH 8.0; 150 mM NaCl; 1 mM EDTA; 1% NP-40; 0.5% sodium deoxycholate; 10% of protease) (Roche Diagnostic, Indianapolis, IN, USA) and 10% phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA, USA). The extracts were centrifuged at 14,000× g
at 4 °C for 15 min, and the total protein concentration was estimated by using Bradford reagent (Bio-Rad Laboratories, Inc, Philadelphia, PA, USA). A total of 15 μg of protein was mixed with loading buffer (62.5 mM Tris-HCl pH 6.9; 2.5% SDS (Merck & Co, Kenilworth, NJ, USA), 8.7% glycerol (Merck & Co, Kenilworth, NJ, USA), 0.5 mM EDTA, 2.5% β-mercaptoethanol (Merck & Co, Kenilworth, NJ, USA), and bromophenol blue (Merck & Co, Kenilworth, NJ, USA)), denatured by heating at 95 °C for 5 min and subjected to separation in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel. The proteins were then blotted onto nitrocellulose membrane and blocked in 5% non-fat milk diluted in a mixture of TBST (tris-buffered saline) (150 mM NaCl; 50 mM Tris pH 7.4; 0.1% Tween®
(Sigma Aldrich, Allentown, PA, USA) for one hour at room temperature, followed by incubation with primary antibody (Table S7
) overnight in TBST buffer with 5% bovine serum albumin (BSA). The membranes were washed and incubated with peroxidase conjugated-secondary antibody. The bands were visualized using ECL (enhanced chemiluminescent) detection reagents (GE Healthcare, Chicago, IL, USA). Densitometric quantification was performed by ImageJ software (https://imagej.net/ImageJ
). The graphics express the protein levels in the relative amount of the membrane stained with Ponceau S solution (Sigma Aldrich-Merck, Germany).
Transfected TPC1 cells (1.5 × 105) were seeded on coverslips in a 24-well culture plate for 24 h. Cells were treated with cisplatin (10 μM) for 1 h, fixed with 4% formaldehyde methanol-free diluted in 1× PBS for 20 min at room temperature. Cells were washed three times with 1× PBS, and the buffer (1× PBS + 0.5% Triton X-100) was added for five minutes, following a blocking step for one hour in 5% BSA in 1× PBS. For the detection of DNA damage, coverslips were incubated with primary antibody Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAB (Alexa Fluor® 647 Conjugate) (#9720, Cell Signaling, Danvers, MA, USA) and labeled with DAPI (4’,6-diamidino-2-phenylindole) (Molecular Probes, Thermo Fisher Scientific, Whaltam, MA, USA). Coverslips were washed three times with 1× PBS and were mounted onto microscope slides with FluorSave™ (Merck Millipore, Calbiochem, Burlington, MA, USA). The analysis was performed using the Fluoview FV10i (Olympus, Center Valley, PA, USA) counting an average of 700 cells/field in three fields/coverslips. At least 2000 cells were quantified in each condition.
All data generated or analyzed during this study are included in this manuscript and in the Supplementary Materials
4.7. Copy Number Alteration and DNA Methylation from Publicly Available Data
We interrogated publicly available databases to assess copy number alteration and DNA methylation as a potential mechanism involved in MUS81
gene expression. Invasive ductal breast carcinomas (BC) (N
= 678) plus adjacent normal tissues (N
= 85), and papillary thyroid carcinoma (PTC) (N
= 188) plus adjacent normal tissues (N
= 21) were obtained from the UCSC Xena (https://xena.ucsc.edu
) and cBioPortal (https://www.cbioportal.org
) (both accessed on 8 April 2020). Only samples with available copy number alteration, gene expression, and methylation status were selected. Homozygous deletion, single copy deletion, diploid normal copy, low-level copy number amplification, and high-level copy number amplification were identified by the GISTIC algorithm, which combines the frequency and amplitude of an aberration to rates each segment and apply a permutation test to assess the statistical significance. From TCGA (The Cancer Genome Atlas) level 3, we obtained the RNA-seq data, with values log2(x
+ 1) transformed and normalized and methylation (Illumina450K) with beta values ranging from 0–1. Probes that were shown to be cross-reactive and SNP-affected were filtered out. Data analysis and visualization were conducted with statistical and graphical packages available from R. Furthermore, using the aforementioned TCGA BC and PTC and additional 50 PTC paired with the adjacent tissue [51
], we investigated methylation probes within and outside CpG island sites of the MUS81 region. We evaluated both copy number alteration and methylation in relation to MUS81