Down-regulation of FA-BRCA pathway in the molecular pathogenesis of uterine cervical carcinoma of Indian population: clinical and prognostic implications

doi:10.21203/rs.3.rs-1944207/v1

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Down-regulation of FA-BRCA pathway in the molecular pathogenesis of uterine cervical carcinoma of Indian population: clinical and prognostic implications

https://doi.org/10.21203/rs.3.rs-1944207/v1

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Objective: Our study was aimed to understand the importance of FA-BRCA pathway genes in cervical carcinogenesis and their association with the prognosis of the disease.

Material and methods: we analysed the molecular profiles of the key regulatory genes of FA-BRCA pathway (BRCA1, BRCA2, FANCC, FANCD2) in 109 cervical lesions at different clinical stages and validated in different bioinformatical analysis as well. The results were next correlated with different clinicopathological parameters. Furthermore, the drug tolerance mechanism of the genes was characterized by treating two CACX cell lines (SiHa and HeLa) in presence of the chemotherapeutic drug cisplatin.

Result: Our data showed that the expression pattern (mRNA/Protein) of the genes of FA-BRCA pathway was gradually decreased from normal cervical epithelium to the development of carcinogenesis, also validated in different GEO datasets. Further, in-depth look into the results revealed that genetic (deletion) and epigenetic alterations (promoter methylation) [30 to 55 %] of the genes was strongly correlated with their reduced expression and development of cervical cancer among the patients, resulting in worst 5-year overall survival trend. Incidentally, the prevalence of promoter methylation in both plasma and respective tumour DNA of invasive cervical carcinoma patients implicated its prognostic importance and association with disease recurrence in this study. In continuation of that, our in-vitro study revealed that cisplatin could upregulate the FA-BRCA pathway genes gradually with increasing drug concentrations in the CACX cell lines through promoter hypomethylation due to reduced expression of DNMT1, indicating the intrinsic mechanism of drug tolerance of residual tumour cells of the disease.

Conclusion: Our data showed that the inactivation of FA-BRCA pathway was associated with the development and prognosis of CACX. Up-regulation of the pathway genes in presence of cisplatin in the CACX cell lines suggested a plausible mechanism of non-responsiveness to the therapy.

Cervical Carcinoma

FA-BRCA pathway

Cisplatin

Disease-recurrence

Promoter hypomethylation

DNMT1

Down-regulation of FA-BRCA proteins emphasizes the genomic instability in the pathogenesis of tumour development
Prevalence of plasma promoter methylation at the advanced CACX stage implies its prognostic importance
Patients having higher alterations of the genes result in poor patient survival
Over-expression of FA-BRCA proteins in residual drug-treated cancer cells develops disease recurrence

Uterine cervical carcinoma (CACX) is the third most commonly diagnosed cancer and also the fourth leading cause of cancer death in females [Arbyn,2011]. The Indian subcontinent also accounted for the second highest reported cancer according to its incidence rate (20.2 in 100,000) [IARC/ICO Summary report, 2021]. Along with several other etiological factors, persistent infection by the high-risk human papilloma (hr-HPV) virus (HPV16, 18, 33 etc.) is said to be the major cause of tumour development [Burd, 2003]. Several studies revealed that hr-HPV initially infects through the squamo-columnar junction of normal cervical epithelium and activates the transformation of undifferentiated basal-parabasal layers of the epithelium in presence of viral oncoproteins specially E6 and E7, resulting in the gradual development of premalignant cervical intraepithelial neoplasia (CIN) and invasive carcinoma (CACX) [Pal and Kundu et al, 2020; Kines et al, 2009; Lambert, 1991]. Several cytogenetic and whole genome sequencing analysis have identified many important cellular pathways to be associated with this disease [Pérez et al, 2007; Campos et al, 2016; Rao et al, 2004]. Among those pathways, DNA Damage Repair pathway (DDR) is of considerable importance due to its role in maintaining the host genomic integrity and stability [Spriggs & Laimins, 2017]. Out of those DDR pathways, the FA-BRCA pathway is considered to be the major one due to frequent alterations of its key regulatory genes BRCA1, BRCA2, FANCC and FANCD2 in many human cancers including lung cancer, urinary bladder cancer, breast cancer and cervical cancer (CACX) as well [Liu et al,2015; Basu et al, 2020; Dasgupta et al, 2017; Latiman et al, 2019]. In this pathway, formation of Fanconi anemia (FA) core complex is an important phenomenon, which activates FANCD2 and subsequently recruits BRCA1 and BRCA2 to the DNA damage site to perform error-free repair via homologous recombination [Fang et al, 2020]. Hence, alteration in any one of the genes can impair the whole pathway and develop tumorigenesis. Different studies showed that around 26–45% of genetic alterations of BRCA1-BRCA2 have also been reported in previous studies of uterine carcinoma [Bruchim et al, 2010; Alvarez et al, 2005]. Additionally, more than 35% of CACX patients showed reduced expression of either FANCC or BRCA1 genes due to promoter methylation in previously published reports of cervical carcinoma (CACX) [Narayan et al, 2004]. On the other hand, increasing evidence of viral oncoproteins like E6/E7 plays a crucial role for impairing the function of FANCD2 in the development and progression of CACX [Khanal & Galloway, 2019]. Thus, to understand the importance of the FA-BRCA pathway in the development of CACX it is pertinent to analyse the molecular profiles (expression/deletion/promoter methylation) of the key regulatory genes BRCA1, BRCA2, FANCC, FANCD2 in normal cervical epithelium followed by their changes during development of the disease. Apart from the pathogenesis of the disease, the knowledge of alterations of the FA-BRCA pathway genes during therapy of CACX patients has immense clinical importance. In CACX, generally, cisplatin-based concurrent chemoradiotherapy (CCRT) is usually given to the patients. Some previous studies have already shown that induction of the FA-BRCA pathway was associated with non-responsiveness to therapy in carcinoma patients [Li & Zhang, 2013; Prasad et al, 2017.]. It seems that epigenetic events play an important role in regulation to acquire drug tolerance in the system [Sharma et al, 2010; Glasspool et al, 2006]. However, the detailed molecular association of the key regulatory genes of the FA-BRCA pathway with the failure of cisplatin therapy is not well established.

So, in the given backdrop of information lacunae, the present study was made initially to analyse the molecular profile (copy number variation/promoter methylation/expression) of key regulatory genes of the FA-BRCA pathway in the normal cervical epithelium, and primary cervical lesions at different clinical stages followed by their correlations with different clinico-pathological parameters for better understanding of the role of this pathway in disease development. Secondly, the prevalence of promoter methylation of the FA-BRCA genes in circulating tumour cells (CTCS) of plasma DNA of respective CACX patients was evaluated to check their prognostic importance as well. At last, to find out the importance of the genes in the recurrence of the disease, their molecular profiles (methylation/expression) were evaluated in residual tumour cells of the CACX cell lines after treatment with cisplatin at different concentrations.

Our data revealed that frequent inactivation (deletion and promoter methylation) of FA-BRCA pathway genes were associated with the development and prognosis of the disease. In addition, over expression of those genes due to reduced promoter methylation frequency was observed in cisplatin-tolerant cervical cancer cells of our in-vitro analysis implying their role in drug tolerance.

Collection of clinical samples:

Freshly operated/biopsy samples of 109 cervical lesions along with 5 ml EDTA treated blood of respective patients were collected from the hospital section of Chittaranjan National Cancer Institute (CNCI), Kolkata, India. In addition, cervical epithelium tissues (N= 35) were collected as control who underwent hysterectomy due to other gynaecological reasons. Informed consent from the patients and approval from the research ethics Committee of the Institute were obtained before sample collection. Clinico-pathological characteristics of the patients were also recorded in Table 1. A Part of the freshly operated samples was paraffin embedded after formalin fixation for immunohistochemical analysis. Other part of the samples was frozen immediately at -80°C for DNA and RNA isolation. Additionally, PBL from 36 invasive carcinoma samples were collected, and plasma was separated by short centrifugation and stored at -80°C for further analysis. The tumours were graded and staged according to the International Federation of Gynaecology and Obstetrics (FIGO) classification. The detailed schematic of the samples utilized for various analyses has been given in Table S1.

Cell Culture:

The CACX cell lines (SiHa and HeLa), purchased from National Centre for Cell Science, Pune, India, were cultured in DMEM (Dulbecco’s Modified Eagle Medium) (Gibco #10566016) with 10% fetal bovine serum albumin (Gibco #26140087) and 1% Pen-Strep according to the manufacturer’s protocol. The cells were harvested in a 37ºC incubator with 5% CO₂, as per standard protocol.

Bioinformatical analysis:

Four datasets viz. the GSE6722, GSE39001, GSE6791 and GSE9750 were selected from the GEO NCBI database. Databases used both normal as well as cervical lesions of different clinical stages for the comparison of mRNA expression analysis. The samples were analysed with GEO2R to find out the differentially expressed genes. The result was plotted as volcano plots using GraphPad Prism 8.0. The blue dots represent significant as well as differentially under-expressed [log (fold change) < 0 value and p < 0.05] genes and the red dots represent significant and differentially overexpressed genes [log (fold change) > 0 and p < 0.05] in tumour samples in comparison to normal tissues. The heat map was generated using the log (fold change) of each of the genes, obtained from the four data sets. Next, the Cancer Genome Atlas cervical carcinoma (TCGA-CACX) dataset was assessed for the promoter methylation profile analysis of the key regulatory genes of FA-BRCA pathway in both cervical epithelium and primary cervical lesions by UCSC XENA browser: https://xena.ucsc.edu.

4. DNA extraction from samples:

A. The contaminant normal cells present in cervical lesions of tissue cryosections (5µM) were removed by laser capture micro-dissection (LCM) microscope (Zeiss Palm/Apotome, Germany) for tumour cells enrichment (>80%).

B. In addition, the different layers (basal–parabasal/spinous layers) of serial cryo-sections (5µM) of normal cervical epithelium were also separated by the LCM technique and collected into a micro-centrifuge tube [Fig S1]. DNA from the micro-dissected samples, PBL, plasma (isolated from PBL) and the CACX cell lines (SiHa and HeLa) were isolated by Proteinase-K digestion followed by the phenol-chloroform method [Chakraborty et al, 2022].

5. Detection of HPV in cervical lesions:

A. HPV infection in the samples was detected by polymerase chain reaction (PCR) using primers (MY09 and MY11) from the consensus L1 region followed by typing of HPV16/18 using type-specific primers in the L1-positive samples [Sambrook et al, 1989; Singh et al, 2006]. The primer details are given in Table S2A.

6. Expression Analysis:

A. mRNA expression analysis:

The mRNA expression of the genes (BRCA1, BRCA2, FANCC and FANCD2) was analysed in primary cervical lesions (N=44) along with normal cervical epithelium (N=15) and the CACX cell lines. Total RNA was isolated using TRIzol reagent followed by cDNA synthesis using Superscript III (Invitrogen, USA) reverse transcriptase. Quantitative real-time PCR (qRT-PCR) of the candidate genes was performed using a power SYBR green assay (Applied Biosystems, USA) with beta-2-microglobulin (B2M) as endogenous control [Sambrook et al, 2006]. The primer sequences are listed in Table S2B. The dCt value was obtained using the formula: dCt= [(Ct value) target gene-(Ct value) B2M, [Chakraborty et al, 2022]

B. Immunohistochemical characterization of proteins:

The protein expression pattern of BRCA1, BRCA2, FANCC and FANCD2 was analysed by IHC in the normal cervical epithelium (n = 22), CIN (n = 6) and CACX samples (n = 38) according to the standard procedure as described by Ghosh et al,2008. The list of primary and secondary antibodies used are listed in Table S2C. 3–3′ diaminobenzidine (DAB, sc-24982) was used as the chromogen. A negative control for determination of specificity of the primary antibody was used, where the slides were incubated with PBS, instead of primary antibody. The staining intensity (1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells (<1 cell = 0, 1-20 cells = 1, 20-50 cells = 2, 50-80 cells= 3, >80 cells=4) were detected by two independent observers and by combining the two scores, final evaluation of expression was done (0–2 = low, 3–5 = intermediate, 6–7 = high) according to Perrone et al,2006.

7. Promoter methylation analysis:

Promoter methylation analysis of the key regulatory genes was performed separately in the composite (total) normal cervical epithelium (N = 35) later followed in micro-dissected basal-parabasal/spinous layer of the respective epithelium [Fig S1] and primary cervical lesions (n =109), plasma DNA of CACX samples (n=36) and the CACX cell lines by PCR-based methylation-sensitive restriction analysis (MSRA) using the restriction enzyme Hpa-II (Promega, Fitchburg, WI)) [Islam et al, 2020]. Mock digestion was done for each sample devoid of the enzyme. The β-3A adaptin gene (K1) and RARβ2 exon-1 (K2) were used as digestion and integrity controls respectively. The location of the primers and sequences are represented in Table S2D.

The promoter methylation frequency of the genes was also quantified by real-time PCR using a power SYBR Green assay (Applied Biosystems, USA). The percentage of hypomethylation was calculated using the following formula: (Ct [digested]/Ct [undigested] ×100) –100), [Chakraborty et al, 2022].

[Digested DNA = sample treated with Hpa-II enzyme, Undigested DNA = respective sample not treated with Hpa-II enzyme, presuming the undigested DNA as 100% methylated. Higher percentage of hypomethylation indicates lower frequency of methylation in the sample and vice versa].

Microsatellite-based deletion mapping:

Deletion analysis of the key regulatory genes was done by microsatellite-based deletion mapping in primary cervical lesions (N=109). The detailed information about the microsatellite markers is given in Table S2E. For the control, the DNA isolated from the PBL of the cancer patients was used. In deletion mapping, a standard PCR was carried out using a γ[p32] ATP labeled forward primer and T4- polynucleotide kinase (Promega), followed by amplifying the genomic DNA and finally performed electrophoresis on 8% denaturing polyacrylamide gel containing 8M urea. Loss of heterozygosity (LOH) was scored by densitometric scanning (Bio-Rad GS-800, USA) to determine the loss of one allele in the tumour compared to the same allele in normal (PBL) [Chakraborty et al, 2022]. Microsatellite size alteration (MA) was detected as a shift in one (MA-I) allele compared with their normal alleles.

Treatment of chemotherapeutic drug (Cisplatin) on CACX cell lines:

The effect of antitumour drug- cisplatin (Selleckchem, product id-S1166) on SiHa and HeLa cell lines was analysed by dye sulforhodamine B (SRB) to do the drug kinetics and the dose selection was done according to the standard protocol [Sur et al, 2016]. For cytotoxic assessment of Cisplatin, 5000 cells were seeded in each well of 96- well plate and after 48 hours of Cisplatin treatment, cells only adhered to the plates were fixed with 10% (wt/vol) trichloroacetic acid and stained for 30 min with 0.04% (wt/vol) SRB solution, after which the dye was removed by washing repeatedly with 1% (vol/vol) acetic acid. The protein-bound dye was dissolved in 10 mM Tris base solution (pH 10.5) Finally, the optical density of each well was determined at 510 nm using a microplate reader [Vichai, & Kirtikara, 2006] and IC30-IC50 and IC70 concentrations of the drug were determined (unpublished data).

A. Effect of drug on molecular profile of the genes:

For further experiments as mentioned below, SiHa and HeLa cells (approximately 10⁷cells) were treated with IC30, IC50 and IC70 concentrations of cisplatin drug for 48 hrs. Cells without cisplatin treatment had been raised simultaneously and used as control.

i. Expression (mRNA/protein) analysis:

These treated and untreated (control) cells were subjected to expression (RNA/protein) analysis. Then isolated RNA from both of the cell lines (control and treated) were subjected to real-time quantification of the genes (qRT-PCR) as described earlier using the following formula:

ddCt = [ (Ct)target gene − (Ct)B2M] (treatment) − [ (Ct)target gene − (Ct)B2M] (control)

The value of fold change (ddCt) ≤0.66 and (ddCt) ≥1.5 was considered as under-expression and over-expression respectively [Chakraborty et al, 2022].

The protein expression of the genes after cisplatin treatment in the CACX cell lines was analysed by western-blot analysis [Islam et al, 2020]. Whole-cell protein was extracted by sonication with RIPA buffer and quantified by Bradford reagent. About 50 ug of proteins of the samples were run in 10-12% SDS polyacrylamide gels, transferred to polyvinylidene fluoride (PVDF) membrane and incubated with the primary and secondary antibodies for desired proteins, listed in Table S2C. The membranes were later developed with luminol (sc-2048; Santa Cruz Biotechnology). All the experiments were run in three replicates. The band intensities of the target proteins were plotted on a graph after being normalized by band intensities of loading control α- tubulin and calculated using Image-J software.

For immunocytochemistry, a monolayer of cells was grown on sterile coverslips and fixed with methanol at −20 °C. Then the cells were reacted with the desired amount of primary antibody after permeabilization with 0.5% Triton X-100 and blocking with 5% BSA. After washing, the coverslips were incubated with fluorescein isothiocyanate (FITC)-conjugated corresponding secondary antibody at 1:500 dilutions and mounted with glycerol after thorough washing. The list of primary and secondary antibodies used for the experiment are listed in Table S2C. Imaging of the coverslip was performed in a fluorescence microscope (Leica DM4000 B, Germany) [Islam et al, 2020]

ii. Promoter methylation analysis:

For promoter methylation analysis, DNA from both cisplatin-treated/untreated CACX cell lines were subjected for restriction enzyme-based digestion followed by quantitative analysis, the percentage of hypo-methylation was calculated as described earlier [Chakraborty et al, 2022].

Statistical analysis:

Fisher’s exact test was used to determine different clinico-pathological associations with alterations (genetic/epigenetic) of the cervical lesions. Z-score was applied as per requirement, for determining the level of significance between the groups. Spearman Correlation test was done to analyse the correlation coefficient between two groups. Survival curves were obtained according to Kaplan–Meier method. Overall survival (OS) was measured from the date of surgery to the date of most recent follow-up or death (up to 5 years). The statistical analyses were performed using statistical programs Epi Info 6.04, SPSS 10.0 (SPSS, Chicago, IL). A t-test was performed to compare the expression (mRNA/protein) and percentages of hypomethylation in CACX cells between untreated and drug-treated samples. All statistical tests were two-sided and considered significant at a probability value, p < 0.05.

HPV prevalence in primary cervical lesions:

HPV prevalence was found in 88.8% (24/27) and 98.78% (81/82) of CIN and CACX samples respectively [Fig S2]. Out of HPV positive CIN samples, major samples were HPV 16 positive (87.5%; 21/24) followed by HPV 18 (8.33%; 2/24) and HPV 16/18 mixed positive (4.16%; 1/24). Among the HPV positive CACX samples, 82.71% (67/81) and 11.11% (9/81) of the samples were HPV 16 and HPV 18 positive respectively and the remaining 6.17% (5/81) were both HPV 16/18 mixed positive [Fig S2].

2. Expression profile analysis of FA-BRCA pathway genes:

A. In publicly available GEO dataset:

Four publicly available datasets viz. the GSE6722, GSE39001, GSE6791 and GSE9750 from GEO NCBI database were evaluated for expression microarray analysis. Where the different stages of CACX samples were compared with respect to normal samples, in order to understand the role of this pathway in the progression of this disease. In tumour samples, significant and differential downregulation of BRCA1 and BRCA2 was observed in majority of the datasets [Fig. 1; A-B]. Whereas FANCC and FANCD2 genes were found to be significantly down-regulated in tumour samples of GSE67522/GSE39001 and GSE39001/GSE6791 datasets respectively [Fig. 1; A-B]. This indicates that these key regulatory genes may play an important role in the early steps of carcinogenesis from normal to tumour tissues.

B. mRNA expression profile in the samples:

The mRNA expression of the key regulatory genes of the FA-BRCA pathway was comparable (BRCA1/FANCD2) or slightly decreased (BRCA2/FANCC) in CIN followed by gradual down-regulation with progression of the disease [Fig. 1; C-F]. Interestingly, significant down-regulation of FANCC (p= 0.019) was found in stage-I/II samples, whereas, BRCA1 and FANCD2 expression were significantly (p= 0.036, 0.007) reduced in stage-III/IV compared to stage-I/II. Therefore, under expression of key regulatory genes of FA-BRCA pathway, as found from the GEO datasets, was well in concordance with our sample pool. The expression profile of the genes in the CACX cell lines was also checked and seemed to be comparable with the tumour samples [Fig. 1; C-F].

C. Immunohistochemical expression profile in the samples:

The mRNA expression pattern was next correlated with the protein expression of the genes by immunohistochemistry [Fig. 2; A-D]. The prevalence of high/medium (H/M) predominant nuclear expression of BRCA1, BRCA2 and FANCD2 proteins was high in basal/parabasal layers of cervical epithelium with 81.8%, 86.36% and 68.18% respectively followed by their gradual decrease in spinous layer (63.6%, 68.18% and 40.90%) [Fig. 2; A-B, D]. Whereas, high frequency (86.36%) of FANCC expression (H/M) was seen throughout the entire cervical epithelium [Fig. 2; C].

On contrary to Normal cervical epithelium, the nuclear expression of BRCA1 protein was gradually decreased from CIN (66.60%) to stage-I/II (57.69%) and significantly reduced (p=0.018) in later invasive stage (stage-III/IV) (16.66%) [Fig. 2; A]. Interestingly, significant downregulation (p=0.030) of FANCC was preferably noticed in the early invasive stage (stage-I/II) (34.61%) of cervical lesions and maintained in stage-III/IV as well (25%) [Fig. 2; C], this phenomenon may insight that role of downregulation of the key regulatory genes through the different stages of the disease is needed according to the need of tumorigenesis. Furthermore, similar trend of comparable protein expression of both BRCA2 and FANCD2 was observed in both CIN and stage-I/II (around 50%) and reduced in stage- III/IV (around 17%), [Fig. 2; B, D]. Apart from this, protein expression pattern of all the key regulatory genes showed strong concordance with their respective mRNA expression as well [Table S3A].

Among the invasive lesions, around 59.09% of the samples showed co-downregulation of more than one protein of FA-BRCA pathway, signifying the cumulative role of down-regulation of these genes in the development of CACX [Fig. 2; E].

3. Promoter methylation analysis of FA-BRCA pathway genes:

A. In publicly available TCGA-CACX dataset:

In the TCGA cohort, promoter methylation is represented in the form of β-values which ranges from 0 (unmethylated) to 1 (fully methylated). The promoter methylation frequencies of BRCA1, BRCA2 and FANCC were significantly (p= 0.0004, 0.003, 0.004) high in primary tumour compared to normal tissue. On the other hand, FANCD2 showed very slight change in promoter methylation frequency in tumour tissue compared to normal. [Fig 3; A].

B. Qualitative promoter methylation profile in the samples:

The TCGA dataset could not explain protein expression levels of the genes in different layers of normal cervical epithelium. So, we tried to address this problem by undertaking promoter methylation analysis in composite (total), laser-guided micro-dissected basal-parabasal (stem cell rich, undifferentiated cells) and spinous layers (more terminally differentiated cells) of normal epithelium and in cervical lesions. The strategies of MSRA have been shown in Fig S1.

Infrequent promoter methylation of the FA-BRCA genes in normal cervical epithelium (total) was seen in the following order- FANCD2 (17.14%)>BRCA1-FANCC (14.28%) and > BRCA2 (11.42%) [Fig 3; B-E (i-ii)]. However, in respective micro-dissected samples, the methylation frequencies of BRCA1, BRCA2 and FANCD2 were low in basal-parabasal layer with 11.42%, 8.75% and 11.42% respectively than the spinous layer (17.14%, 11.42% and 20.00%) [Fig 3; B-C, E (i-ii)]. Unlike these genes, uniform promoter methylation of FANCC was found in both the layers (14.28%) of normal cervical epithelium [Fig 3; D (i-ii)].

In cervical lesions, promoter methylation of all the genes was gradually increased from CIN to subsequent clinical stages compared to the basal-parabasal layer of normal tissue [Fig 3; B-D (i-ii)]. Noticeably, the methylation frequencies of FANCC and BRCA2/FANCD2 genes were significantly high (p=0.027-0.039) in early (stage-I/II) and later (stage-III/IV) invasive lesions respectively [Fig 3; C-E (i-ii)]. The promoter methylation pattern of all the key regulatory genes showed strong concordance with their respective protein expression as well [Table S3B]. This event indicates the plausible role of promoter methylation of the genes for down-regulation of respective protein expressions during the progression of the disease.

C. Quantitative promoter methylation profile in the samples:

The methylation pattern of the genes was further validated by quantitative MSRA (q-MSRA), as it gives us an idea of the extent of promoter methylation percentage in each sample [24]. Dot-plot obtained from the q-MSRA data revealed the sample-wise details of the changes in promoter methylation events during tumorigenesis from normal cervical epithelium (basal-parabasal and spinous layer). In q-MSRA analysis, the result showed that hypo-methylation frequencies of FA-BRCA pathway genes in basal-parabasal and spinous layers showed concordance with their qualitative MSRA analysis followed by a gradual decrease in CIN and subsequent clinical stages [Fig 3; F (i-iv)]. [lower the percentage of hypomethylation indicates higher frequency of methylation in the samples].

4. Deletion analysis of FA-BRCA pathway genes:

To evaluate the molecular mechanism behind the under expression of the genes, deletion analysis was performed. Deletion frequency of BRCA1 and BRCA2 was gradually increased from CIN (33.33%, 25.92%) to stage-I/II (46.15%, 38.46%) and became significantly (p= 0.024, 0.030) high at stage-III/IV (73.33%, 63.33%) [Fig 4; A-B (i-ii)]. However, infrequent deletion frequency of FANCC in 14.81% of CIN samples became significantly (p= 0.043) increased in early invasive stages (36.53%) and remained comparable in stage-III/IV (40%) as well. Compare to FANCC, deletion frequency of FANCD2 was comparatively high on pre-malignment CIN samples (29.63%) and gradually increased in stage-I/II (48.07%) and stage-III/IV (56.66%) [Fig 4; C-D (i-ii)]. In addition to deletion, infrequent MA of BRCA1 and BRCA2 was noticed in around 11.11% and 7.40% of CIN samples followed by their gradual increase in stage-I/II (17.37%, 13.46%) and became significant (p= 0.048, 0.032) at stage-III/IV (36.66%, 33.33%) [Fig 4; A-B (i-ii)]. This phenomenon insight the role of genomic instability occurred due to alteration of any of the genes of FA-BRCA pathway actually induces the development of early steps of carcinogenesis.

5. Clinico-pathological association with alterations of FA-BRCA pathway genes:

In totality, overall alterations (either methylation and/or deletion) of the genes of CIN samples were seen in the following order- FANCD2 (55.55%)> BRCA2 (44.44%)> BRCA1/FANCC (37.03%) with gradual increase with progression of the disease [Fig 4; E (i-iv)]. Interestingly, overall alterations in FANCC and BRCA2 were significantly high (p= 0.0009, 0.002) in early and later invasive stages respectively [Fig 4; E (ii-iii)]. This event is signifying that alteration of any of the alleles may actually play an anchoring role in regulating the reduced expression of these genes.

On the other hand, co-alterations of the genes (both methylation and deletion) were also gradually increased with tumour development [Fig 4; E (i-iv)]. The result showed that the co-alteration frequencies of both BRCA1 and FANCD2 were significantly raised (p= 0.030, 0.012) at stage-III/IV samples.

In 5-year follow-up study (Kaplan-Meier analysis) revealed patients with BRCA1-deletion in tumours had poor patient outcomes [Fig 5; A]. In addition, patients having overall alterations (Methylation and/or Deletion) of either one of the key regulatory genes like- BRCA1/BRCA2/FANCC/FANCD2 showed the worst prognosis among patients [Fig 5; B-E]. Similarly, patients with co-alteration of BRCA1-BRCA2-FANCC-FANCD2 together had poor survival, suggesting the prognostic importance of the FA-BRCA pathway genes with disease outcome [Fig 5; F].

6. Prevalence of promoter methylation of FA-BRCA pathway genes in plasma DNA of CACX patients and its association with survival:

To evaluate the usefulness of promoter methylation of the FA-BRCA pathway genes in plasma DNA, 36 primary cervical lesions were analysed. Details of therapy (CCRT therapy) and the survival status of the respective patients were also recorded in their follow-up data. In our study, methylation frequencies of the genes in plasma DNA derived from circulating tumour cells (CTCs) were in the following order- BRCA1-BRCA2 (16.66%)> FANCD2 (13.88%)> FANCC (11.11%) [Fig 6; A-D]. Noticeably, it was evident that the presence of promoter methylation of the respective genes was more prevalent in later invasive lesions (stage-III/IV) compare to early stage (stage- II). Prevalence of promoter methylation of the genes (BRCA1/FANCC/FANCD2) in plasma DNA also showed significant (p= 0.029- 0.042) concordance of their methylation frequency in respective primary tumours. [Fig 6; E].

Interestingly, patients having methylation of any one of the genes in plasma showed poor patient outcome even after the therapeutic intervention [Fig 6; E].

7. Effect of cisplatin-treatment in the molecular profile of FA-BRCA pathway genes in CACX cell lines:

After analysing the poor patient survival due to the non-responsiveness of the patients to the treatment in our follow-up data as discussed previously [Fig 6], our aim was to understand the molecular mechanism of the FA-BRCA pathway genes in disease recurrence among the CACX patients. For this purpose, the CACX cell lines were treated with different concentrations of cisplatin and then the molecular profile (expression/methylation profile) of the target genes was evaluated in our in-vitro study.

A. Cisplatin sensitivity of the CACX cell lines:

The CACX cell lines showed different sensitivity towards cisplatin treatment. Hela cells were seen to be more sensitive to cisplatin than SiHa cells [Unpublished Data].

B. Effect of cisplatin on the expression of the genes in CACX cell lines:

After treating the cell lines with different concentrations of cisplatin for 48 hrs, mRNA expression of FA-BRCA pathway genes was analysed. It was evident that mRNA expression was slightly increased at IC30 and became significantly high at a gradual increment of the drug concentrations of IC50 and IC70 in both of the cell lines [Fig 7; A (i-iv)].

In western blot analysis, the protein expression also gradually increased with the increasing drug concentrations and predominantly raised at IC50 and IC70 of cisplatin in both of the cell lines, showing concordance with their mRNA expression in both the cell lines [Fig 7; B (i-v)], indicating transcriptional activation of the genes after cisplatin treatment in the cell lines.

C. Effect of cisplatin on promoter methylation of the genes in CACX cell lines:

To understand the molecular mechanism behind the upregulation of the FA-BRCA pathway genes in cisplatin treated cells, their promoter methylation status was analysed by q-MSRA. The hypomethylation frequency of the genes after cisplatin treatment in the cell lines was gradually increased (higher the value of hypomethylation percentage indicates lower the promoter methylation and vice versa) with drug concentrations and became significantly high at IC50 and IC70 concentrations, showing concordance with expression of the genes [Fig 7; C (i-v)].

8. Effect of cisplatin on DNMT1 expression in CACX cell lines:

To find out the plausible reason for reduced promoter methylation of the genes in cisplatin-treated cell lines, DNMT1 expression (both mRNA and protein) was analysed, due to its importance in the maintenance of tissue-specific methylation [Klein et al, 2011]. It was evident that m-RNA expression of DNMT1 was prominently reduced at IC50 and IC70 concentration of cisplatin in SiHa and only at IC70 in HeLa cell line respectively [Fig 8; A].

In the western blot analysis of DNMT1, gradual decrease in protein expression of DNMT1 was seen with increasing drug concentrations and showed concordance with its mRNA expression [Fig 8; B-C].

9. Effect of cisplatin on immunocytochemical analysis of ϒ-H2X foci in CACX cells:

For functional evaluation of the up-regulated FA-BRCA pathway genes in cisplatin-treated CACX cell lines, the ϒ-H2X nuclear foci formation was analysed after treatment with different concentrations of cisplatin for 48 hrs in CACX cell lines [Fig S3; A-B]. As ϒ-H2X is an important marker of DNA damage [12], our result revealed that there was an increase in the number of ϒ-H2X foci at IC30 (32-37 foci/cell) of the drug compared to untreated control followed by a gradual decrease at IC50 (21-27 foci/cell) and IC70 (12-15 foci/cell) concentrations [Fig S3; C], indicating activation of the DNA repair mechanism in the residual tumour cells in the system.

In this study, we aimed to get a better understanding of the role (expression/promoter methylation /deletion) of the key regulatory genes of FA-BRCA pathway during the development of cervical carcinoma along with their association with different clinico-pathological factors. In addition, modulation of the genes in residual cisplatin tolerant CACX cell lines was also analyzed to understand their clinical importance. In bioinformatical analysis, all four datasets showed differential reduced expression of BRCA1 and BRCA2 along with down-regulation of FANCC and FANCD2, found in only two datasets [Fig. 1]. This data was further validated in our sample pool of Indian patients and obtained similar result for the genes [Fig. 1]. The bioinformatical analysis although gave us an overall idea of the expression pattern of the key regulatory genes of this pathway but, it was not possible to describe the events which could lead to the changes in the expression of the respective genes from different layers of normal cervical epithelium to cervical carcinoma. So, we next undertook protein expression analysis through immunohistochemistry for the key regulatory genes.

Comparatively high nuclear expression of majority of the proteins found in basal-parabasal layers of cervical epithelium than spinous layers were also concordant with their methylation pattern [Fig. 2, 3]. Such a profile of the key regulators of FA-BRCA pathway can be considered as the expression signature of the normal cervical epithelium for maintenance of homeostasis of genomic stability [Paulsen et al,2009; Bhatia et al,2017]. While during tumorigenesis, reduced expression of the genes was observed in CIN samples followed by significant decrease (p = 0.018–0.030) in invasive carcinoma stages emphasising frequent chromosomal aberration as a key event in development of disease. This was further indicated by co-down regulation (59.09%) of the genes in majority of the primary cervical lesions [Fig. 2]. A similar phenomenon has also been reported in others cancers like lung cancer, urinary bladder cancer, breast cancer and multiple myeloma etc. [Liu et al,2015; Basu et al, 2020; Dasgupta et al, 2017; Yarde et al, 2009].

To understand the molecular aspect of reduced expression of the genes, we analysed both genetic (deletion) and epigenetic regulation (promoter methylation) as well. Our data suggested that frequent methylation and deletion of the genes particularly in the invasive carcinoma stages of the tumour were the plausible reasons of their reduced expression and provide a selective growth advantage of the tumour [Fig. 3, 4]. Though FA-BRCA pathway was reported to be altered previously in cervical carcinoma [Latiman et al, 2019; Bruchim et al, 2010; Alvarez et al, 2005] but the detailed molecular mechanism of carcinogenesis (both genetic and epigenetic) in same cervical carcinoma patients of Indian population was studied for the first time in this study. In addition to that, co-alteration (both methylation and deletion) of FA-BRCA genes were also gradually increased (p = 0.012–0.030) with progression of the disease suggesting importance of their inactivation in this tumorigenesis [Fig. 4]. This was also reflected in patient’s survival and data suggested that patients having overall alterations (Methylation/ Deletion) of either one or all of the genes had poor disease-free survival [Fig. 5].

Apart from promoter methylation in tumour tissue sample, prevalence of methylation frequency of these genes (11.11%-16.66%) was also found in plasma of some respective CACX patients. Interestingly, it was noticed that retention of promoter methylation of the genes in circulating tumour DNA of plasma among the invasive CACX patients showed the worst prognosis [Fig. 6]. This information could lead to suggest the prognostic importance of the candidate genes for better understanding about the lethality of the disease among the patients.

Apart from the role of FA-BRCA pathway in tumorigenesis of CACX, the molecular profile of the genes was also analysed in our in-vitro study to find out the plausible reason of non-responsiveness to the cisplatin therapy among the patients. Our data revealed that elevated expression (RNA/Protein) profile of FA-BRCA genes along with their reduced promoter methylation was observed at higher concentrations (IC50, IC70) of drug in both of the CACX cell lines [Fig. 7]. This phenomenon indicates that cells are accomplished enough to overcome the cisplatin-induced DNA damage through upregulation of FA-BRCA pathway leading to drug-tolerance within the system [Balacescu et al, 2014]. This was further validated by gradual decrease in the prevalence of ϒ-H2X foci with increasing drug concentrations. [Fig S3]. It seems that the hypomethylation of the FA-BRCA pathway genes in the presence of the drug might be due to the inhibition/ reduced expression of the DNA methylating enzymes particularly DNMT1 as seen in our study [Fig. 8], [ Li & Zhang, 2013]. On the other hand, the FACS analysis data also revealed that there was a gradual accumulation of cells at the G2/M phase of the cell cycle (Unpublished data) at higher concentrations (IC50, IC70) of cisplatin-treated CACX cell lines. This might indicate that residual tumour cells were proficient enough to overcome the impact of cisplatin via homologous repair due to extended G2/M phase in the system [Colton et al, 2006].

Thus, this work suggests that frequent inactivation of FA-BRCA pathway genes due to their promoter methylation and/or deletion might play an important role in the molecular pathogenesis of the disease. Interestingly, the prevalence of plasma methylation of the genes could also serve their prognostic and diagnostic importance as well. In addition, over-expression/activation of the FA-BRCA genes was seen to be correlated with failure therapy among the patients, as suggested by our in-vitro study. However, future studies in appropriate model systems are warranted in this regard for a better understanding of their role in tumour development and therapy, where the preliminary findings of this study could serve as a rock bed for future in-depth investigations.

Funding The financial support for this work DST-INSPIRE Fellowship grant Sanction order. No. IF170005/2017, (ii) EU-V dated 17.07.2017) to was provided by Mrs. P. Dutta and to NASI Senior Scientist Platinum Jubilee Fellowship (2020) awarded to Dr. C.K. Panda. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Acknowledgments The authors thank the Director, Chittaranjan National Cancer Institute [CNCI], Kolkata, India for their kind interest in the work. We also thank health professionals and the pathology department for the collection of specimens. Finally, the authors specially thank all the 144 patients who participated in the study.

Compliance with ethical standards:

Ethics statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Written informed consent was obtained from patients, as well as hospital authorities and the institutional ethical committee prior to the study.

Conflict of interest: None declared.

Consent for publication: All the authors of this study agreed to participate in this study.

Availability of data and material: Not applicable

Code availability: Not applicable

Author Contribution: This work was conceptualized and the manuscript was drafted by P. Dutta and Chinmay Kumar Panda. P. Dutta performed the experiments in primary tumour and CACX cell lines while D. Pal, F. Sultana and R. Mandal helped in interpreting the results and did the statistical analysis. R. Mandal provided the tumour samples. A. Roy was helpful in providing pathological guidance and other resources.

Arbyn, M., Castellsague, X., de Sanjose, S., Bruni, L., Saraiya, M., Bray, F. et al. (2011) Worldwide burden of cervical cancer in 2008. Ann. Oncol. 22, 2675–2686 doi:10.1093/annonc/mdr015
IARC/ICO Information Centre on Human Papilloma Virus (HPV) and Related Diseases report in India. Summary Report. 2021
Burd E. M. (2003). Human papillomavirus and cervical cancer. Clinical microbiology reviews, 16(1), 1–17. https://doi.org/10.1128/CMR.16.1.1-17.2003
Pal, A., & Kundu, R. (2020). Human Papillomavirus E6 and E7: The Cervical Cancer Hallmarks and Targets for Therapy. Frontiers in microbiology, 10, 3116. https://doi.org/10.3389/fmicb.2019.03116
Kines, R. C., Thompson, C. D., Lowy, D. R., Schiller, J. T., & Day, P. M. (2009). The initial steps leading to papillomavirus infection occur on the basement membrane prior to cell surface binding. Proceedings of the National Academy of Sciences of the United States of America, 106(48), 20458–20463. https://doi.org/10.1073/pnas.0908502106
Lambert P. F. (1991). Papillomavirus DNA replication. Journal of virology, 65(7), 3417–3420. https://doi.org/10.1128/JVI.65.7.3417-3420.1991
Pérez-Plasencia, C., Vázquez-Ortiz, G., López-Romero, R., Piña-Sanchez, P., Moreno, J., & Salcedo, M. (2007). Genome wide expression analysis in HPV16 cervical cancer: identification of altered metabolic pathways. Infectious agents and cancer, 2, 16. https://doi.org/10.1186/1750-9378-2-16
Campos-Parra, A. D., Padua-Bracho, A., Pedroza-Torres, A., Figueroa-González, G., Fernández-Retana, J., Millan-Catalan, O., Peralta-Zaragoza, O., Cantú de León, D., Herrera, L. A., & Pérez-Plasencia, C. (2016). Comprehensive transcriptome analysis identifies pathways with therapeutic potential in locally advanced cervical cancer. Gynecologic oncology, 143(2), 406–413. https://doi.org/10.1016/j.ygyno.2016.08.327
Rao, P. H., Arias-Pulido, H., Lu, X. Y., Harris, C. P., Vargas, H., Zhang, F. F., Narayan, G., Schneider, A., Terry, M. B., &Murty, V. V. (2004). Chromosomal amplifications, 3q gain and deletions of 2q33-q37 are the frequent genetic changes in cervical carcinoma. BMC Cancer, 4, 5. https://doi.org/10.1186/1471-2407-4-5
Spriggs, C. C., &Laimins, L. A. (2017). Human Papillomavirus and the DNA Damage Response: Exploiting Host Repair Pathways for Viral Replication. Viruses, 9(8), 232. https://doi.org/10.3390/v9080232oi.org/10.4143/crt.2005.37.6.319
Liu, Q., Ghosh, P., Magpayo, N., Testa, M., Tang, S., Gheorghiu, L., Biggs, P., Paganetti, H., Efstathiou, J. A., Lu, H. M., Held, K. D., & Willers, H. (2015). Lung cancer cell line screen links Fanconi anemia/BRCA pathway defects to increased relative biological effectiveness of proton radiation. International journal of radiation oncology, biology, physics, 91(5), 1081–1089. https://doi.org/10.1016/j.ijrobp.2014.12.046
Basu, M., Ghosh, S., Roychowdhury, A., Samadder, S., Das, P., Addya, S., Roy, A., Pal, D. K., Roychoudhury, S., Ghosh, A., & Panda, C. K. (2020). Integrative genomics and pathway analysis identified prevalent FA-BRCA pathway alterations in arsenic-associated urinary bladder carcinoma: Chronic arsenic accumulation in cancer tissues hampers the FA-BRCA pathway. Genomics, 112(6), 5055–5065. https://doi.org/10.1016/j.ygeno.2020.09.012
Dasgupta, H., Mukherjee, N., Islam, S., Bhattacharya, R., Alam, N., Roy, A., Roychoudhury, S., Biswas, J., & Panda, C. K. (2017). Frequent alterations of homologous recombination repair pathway in primary and chemotolerant breast carcinomas: clinical importance. Future oncology (London, England), 13(2), 159–174. https://doi.org/10.2217/fon-2016-0289
Laitman, Y., Michaelson-Cohen, R., Levi, E., Chen-Shtoyerman, R., Reish, O., Josefsberg Ben-Yehoshua, S., Bernstein-Molho, R., Keinan-Boker, L., Rosengarten, O., Silverman, B. G., Perri, T., Korach, J., Mor, P., Ephrat Ben-Baruch, N., Levy Lahad, E., Friedman, E., & Israeli Consortium of Hereditary Breast Cancer (2019). Uterine cancer in Jewish Israeli BRCA1/2 mutation carriers. Cancer, 125(5), 698–703. https://doi.org/10.1002/cncr.31842
Fang, C. B., Wu, H. T., Zhang, M. L., Liu, J., & Zhang, G. J. (2020). Fanconi Anemia Pathway: Mechanisms of Breast Cancer Predisposition Development and Potential Therapeutic Targets. Frontiers in cell and developmental biology, 8, 160. https://doi.org/10.3389/fcell.2020.00160
Bruchim, I., Amichay, K., Kidron, D., Attias, Z., Biron-Shental, T., Drucker, L., Friedman, E., Werner, H., & Fishman, A. (2010). BRCA1/2 germline mutations in Jewish patients with uterine serous carcinoma. International journal of gynecological cancer: official journal of the International Gynecological Cancer Society, 20(7), 1148–1153. https://doi.org/10.1111/IGC.0b013e3181ef622d
Alvarez, S., Diaz-Uriarte, R., Osorio, A., Barroso, A., Melchor, L., Paz, M. F., Honrado, E., Rodríguez, R., Urioste, M., Valle, L., Díez, O., Cigudosa, J. C., Dopazo, J., Esteller, M., & Benitez, J. (2005). A predictor based on the somatic genomic changes of the BRCA1/BRCA2 breast cancer tumors identifies the non-BRCA1/BRCA2 tumors with BRCA1 promoter hypermethylation. Clinical cancer research: an official journal of the American Association for Cancer Research, 11(3), 1146–1153.
Narayan, G., Arias-Pulido, H., Nandula, S. V., Basso, K., Sugirtharaj, D. D., Vargas, H., Mansukhani, M., Villella, J., Meyer, L., Schneider, A., Gissmann, L., Dürst, M., Pothuri, B., &Murty, V. V. (2004). Promoter hypermethylation of FANCF: disruption of Fanconi Anemia-BRCA pathway in cervical cancer. Cancer Research, 64(9), 2994–2997. https://doi.org/10.1158/0008-5472.can-04-0245
Khanal, S., & Galloway, D. A. (2019). High-risk human papillomavirus oncogenes disrupt the Fanconi anemia DNA repair pathway by impairing localization and de-ubiquitination of FancD2. PLoS pathogens, 15(2), e1007442. https://doi.org/10.1371/journal.ppat.1007442
Li, M., Zhang, C. Demethylation of FANCF gene may be a potential treatment through inhibiting the proliferation of cervical cancer. Chin. -Ger. J. Clin. Oncol. 12, 339–342 (2013). https://doi.org/10.1007/s10330-013-1198-7
Prasad, C.B., Prasad, S.B., Yadav, S.S. et al. Olaparib modulates DNA repair efficiency, sensitizes cervical cancer cells to cisplatin and exhibits anti-metastatic property. Sci Rep 7, 12876 (2017). https://doi.org/10.1038/s41598-017-13232-3
Sharma SV, Lee DY, Li B, Quinlan MP, Takahashi F, Maheswaran S, McDermott U, Azizian N, Zou L, Fischbach MA, Wong KK, Brandstetter K, Wittner B, Ramaswamy S, Classon M, Settleman J (2010) A chromatin-mediated drug-tolerant state in cancer cell subpopulations. Cell 141:69–80. https://doi.org/10.1016/j.cell.2010.02.027
Glasspool RM, Teodoridis JM, Brown R (2006) Epigenetics as a mechanism driving polygenic clinical drug resistance. Br J Cancer 94:1087–1092
Chakraborty, B., Basu, M., Mukhopadhyay, D., Alam, N., Ghosh, S., Dutta, S., & Panda, C. K. (2022). Differential promoter usages of PTCH1 and down-regulation of HHIP are associated with HNSCC progression. Pathology, research and practice, 232, 153827. https://doi.org/10.1016/j.prp.2022.153827
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Singh, R.K., Maulik, S., Mitra, S., Mondal, R.K., Basu, P.S., Roychowdhury, S. et al. (2006) Human papillomavirus prevalence in postradiotherapy uterine cervical carcinoma patients: correlation with recurrence of the disease. Int. J. Gynecol. Cancer 16, 1048–1054 doi:10.1111/j.1525-1438.2006.00550.
Sambrook J, Russell DW, Sambrook J. (2006) The Condensed Protocols from Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press:800
Ghosh, S., Ghosh, A., Maiti, G.P., Alam, N., Roy, A., Roy, B. et al. (2008) Alterations of 3p21.31 tumour suppressor genes in head and neck squamous cell carcinoma: correlation with progression and prognosis. Int. J. Cancer 123, 2594–2604 doi:10.1002/ijc.23834.
Perrone, F., Suardi, S., Pastore, E., Casieri, P., Orsenigo, M., Caramuta, S. et al. (2006) Molecular and cytogenetic subgroups of oropharyngeal squamous cell carcinoma. Clin. Cancer Res. 12, 6643–6651 DOI: 10.1158/1078-0432.CCR-06-1759.
Islam, S., Dasgupta, H., Basu, M., Roy, A., Alam, N., Roychoudhury, S., & Panda, C. K. (2020). Downregulation of beta-catenin in chemo-tolerant TNBC through changes in receptor and antagonist profiles of the WNT pathway: Clinical and prognostic implications. Cellular oncology (Dordrecht), 43(4), 725–741. https://doi.org/10.1007/s13402-020-00525-5
Sur, S., Pal, D., Banerjee, K., Mandal, S., Das, A., Roy, A., & Panda, C. K. (2016). Amarogentin regulates self-renewal pathways to restrict liver carcinogenesis in experimental mouse model. Molecular carcinogenesis, 55(7), 1138–1149. https://doi.org/10.1002/mc.22356
Vichai, V., &Kirtikara, K. (2006). Sulforhodamine B colorimetric assay for cytotoxicity screening. Nature protocols, 1(3), 1112–1116. https://doi.org/10.1038/nprot.2006.179
Klein, C. J., Botuyan, M. V., Wu, Y., Ward, C. J., Nicholson, G. A., Hammans, S., Hojo, K., Yamanishi, H., Karpf, A. R., Wallace, D. C., Simon, M., Lander, C., Boardman, L. A., Cunningham, J. M., Smith, G. E., Litchy, W. J., Boes, B., Atkinson, E. J., Middha, S., B Dyck, P. J., … Dyck, P. J. (2011). Mutations in DNMT1 cause hereditary sensory neuropathy with dementia and hearing loss. Nature genetics, 43(6), 595–600. https://doi.org/10.1038/ng.830
Paulsen, R. D., Soni, D. V., Wollman, R., Hahn, A. T., Yee, M. C., Guan, A., Hesley, J. A., Miller, S. C., Cromwell, E. F., Solow-Cordero, D. E., Meyer, T., &Cimprich, K. A. (2009). A genome-wide siRNA screen reveals diverse cellular processes and pathways that mediate genome stability. Molecular cell, 35(2), 228–239. https://doi.org/10.1016/j.molcel.2009.06.021
Bhatia, V., Herrera-Moyano, E., Aguilera, A., & Gómez-González, B. (2017). The Role of Replication-Associated Repair Factors on R-Loops. Genes, 8(7), 171. https://doi.org/10.3390/genes8070171
Yarde, D. N., Oliveira, V., Mathews, L., Wang, X., Villagra, A., Boulware, D., Shain, K. H., Hazlehurst, L. A., Alsina, M., Chen, D. T., Beg, A. A., & Dalton, W. S. (2009). Targeting the Fanconi anemia/BRCA pathway circumvents drug resistance in multiple myeloma. Cancer Research, 69(24), 9367–9375. https://doi.org/10.1158/0008-5472.CAN-09-2616
Balacescu, O., Balacescu, L., Tudoran, O., Todor, N., Rus, M., Buiga, R., Susman, S., Fetica, B., Pop, L., Maja, L., Visan, S., Ordeanu, C., Berindan-Neagoe, I., & Nagy, V. (2014). Gene expression profiling reveals activation of the FA/BRCA pathway in advanced squamous cervical cancer with intrinsic resistance and therapy failure. BMC Cancer, 14, 246. https://doi.org/10.1186/1471-2407-14-246
Colton, S. L., Xu, X. S., Wang, Y. A., & Wang, G. (2006). The involvement of ataxia-telangiectasia mutated protein activation in nucleotide excision repair-facilitated cell survival with cisplatin treatment. The Journal of biological chemistry, 281(37), 27117–27125. https://doi.org/10.1074/jbc.M602826200

TABLE 1: Demography and clinico-pathological features of the cervical samples:

Clinical

features

No of patients

Mean age (yrs)

hr-HPV positivity (HPV 16/18)

% Of positivity

HPV+

HPV-

Cervical epithelium tissue (Control)

Tumour stage:

CIN

CACX-

Stage I/II

Stage III/IV

89%

98%

100%

Tumour grade:

Well

Moderate

Poor

95%

100%

Parity (live births only):

Low parity (0-4)

High parity (≥ 5)

95%

97%

Age of sextual debut:

Early (12-19yr)

Late (> 19 yr)

87.5%

99%

(According to the international federation of gynecology and obstetrics (FIGO) classification)

No competing interests reported.

SupplementaryTables.docx
Supplementary Tables:Table S1: Flow chart of sample utilization.Table S2: Details of primers and antibodies used for experiments.Table S3 A-B: Study of co-relation of FA-BRCA pathway genes in primary cervical lesions.
supplefig1microdissection.tif
Figure S1: A: Pictorial representation of Laser Capture Microdissection procedure and downstream process in unstained cervical epithelium tissue (the red line indicates laser light that separating the basal-parabasal layer from spinous layer of cervical epithelium tissue). B (i-iv): Graphical representation of methylation primer locus of BRCA1, BRCA2, FANCC and FANCD2 - the sideward arrows encompassing the probable primer regions used for promoter methylation analysis containing HpaII (denoted by star) cut sites.
Supplefig2HPV.tif
Figure S2: A: Percentage of High-risk HPV prevalence (HPV16,18) in cervical carcinoma samples of Indian population.
supplefig3gammah2x.tif
Figure S3: A-B: Representative images of immunocytochemical analysis of ϒ -H2X protein in untreated (control) and cisplatin-treated CACX cell lines (SiHa and HeLa). [DAPI: 4’, 6-diamidino-2-phenylindole, FITC: Fluorescein isothiocyanate]. C: Histogram showing average no of nuclear foci count of ϒ-H2X per cell in cisplatin-treated CACX cell lines (SiHa and HeLa).
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Down-regulation of FA-BRCA pathway in the molecular pathogenesis of uterine cervical carcinoma of Indian population: clinical and prognostic implications

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