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ORIGINAL ARTICLE
Year : 2021  |  Volume : 17  |  Issue : 4  |  Page : 1031-1038

Nonhomologous end joining repair pathway molecules as predictive biomarkers for patients with oral squamous cell carcinoma


1 Stem Cell Biology Lab, Department of Cancer Biology, The Gujarat Cancer & Research Institute, Ahmedabad, Gujarat, India
2 Immunohematology Lab, Department of Cancer Biology, The Gujarat Cancer & Research Institute, Ahmedabad, Gujarat, India
3 Tumor Biology Lab, Department of Cancer Biology, The Gujarat Cancer & Research Institute, Ahmedabad, Gujarat, India
4 Department of Surgical Oncology, Gujarat Cancer & Research Institute, Ahmedabad, Gujarat, India
5 Department of Onco-Pathology, Gujarat Cancer & Research Institute, Ahmedabad, Gujarat, India
6 Clinical Carcinogenesis Lab, Department of Cancer Biology, The Gujarat Cancer & Research Institute, Ahmedabad, Gujarat, India

Date of Submission12-Sep-2019
Date of Decision14-Oct-2019
Date of Acceptance19-Dec-2019
Date of Web Publication18-Aug-2021

Correspondence Address:
Trupti I Trivedi
Clinical Carcinogenesis Lab, Department of Cancer Biology, The Gujarat Cancer & Research Institute, Ahmedabad - 380 016, Gujarat
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_582_19

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 > Abstract 


Purpose: Nonhomologous end-joining (NHEJ) is critical for the repair of either pathologic double-strand breaks (DSBs) and/or for the repair of physiologic DSBs created during radiotherapy to kill the tumor cell. Therefore, patients with higher expression of NHEJ repair proteins might develop resistance to ionizing radiation, allowing the disease to recur. As cancer of the oral cavity is a serious health problem globally, the present study aimed to examine the expression of Ku70/80, X-ray repair cross-complementing protein 4 (XRCC4) and DNA ligase IV-core molecules of the NHEJ pathway in patients with oral cancer.
Materials and Methods: Protein expression of Ku70/80, XRCC4, and DNA ligase IV were studied by Immunohistochemistry and mRNA expression of Ku70 and Ku80 were studied using reverse transcription polymerase chain reaction. Data were analyzed statistically using SPSS.
Results: A univariate survival analysis revealed an association of Ku70 mRNA with shorter overall survival (OS). While protein expression of XRCC4 showed an association with reduced relapse-free survival and shorter OS. Multivariate survival analysis demonstrated that XRCC4 and DNA ligase IV are independent prognosticators for predicting adverse disease outcomes.
Conclusion: Strong expression of repair proteins – XRCC4 and DNA ligase IV is associated with unfavorable disease outcome in patients with oral squamous cell carcinoma.

Keywords: DNA ligase IV, Ku70/80, multivariate survival, oral squamous cell carcinoma, X-ray repair cross-complementing protein 4


How to cite this article:
Joshi JS, Vora HH, Ghosh NR, Tankshali RN, Jetly DH, Trivedi TI. Nonhomologous end joining repair pathway molecules as predictive biomarkers for patients with oral squamous cell carcinoma. J Can Res Ther 2021;17:1031-8

How to cite this URL:
Joshi JS, Vora HH, Ghosh NR, Tankshali RN, Jetly DH, Trivedi TI. Nonhomologous end joining repair pathway molecules as predictive biomarkers for patients with oral squamous cell carcinoma. J Can Res Ther [serial online] 2021 [cited 2021 Oct 20];17:1031-8. Available from: https://www.cancerjournal.net/text.asp?2021/17/4/1031/324036




 > Introduction Top


Oral squamous cell carcinoma (OSCC) has a miserable prognosis with an estimated incidence of half a million cases diagnosed per year worldwide.[1] Among them, half of the patients suffer from tumor-related complications because of the heterogeneity and complex behavioral pattern of this malignancy.[2] The 5-year survival rate remains poor at about 15%–50% in spite of the recent advances in diagnostic and therapeutic modalities for the past 30 years.[3],[4],[5],[6] Therefore, earlier detection of oral cancer is becoming ever more important to develop the methods for monitoring disease progression, to increase individual therapeutic efficacy and to offer the best chance of long-term survival. Although in India, an increased incidence of OSCC is owing to the increased prevalence of daily life habits such as chewing betel quid, tobacco, and areca nut, the effect of alcohol cannot be neglected.[7] This tobacco and alcohol consumption may lead to frequent DNA lesions and may damage the genetic codes.[8] Several mutagens found in the tobacco reduces the antioxidant level and induces the oxidative stress, leading to the development of double-strand breaks (DSBs).[9] If such genetic damages are left unrepaired, it may initiate dysregulation in cell cycle and autonomous growth leading to malignant transformation of a particular cell. The repair of such DSBs is critical for maintaining genome stability. Notably, defects in the repair of DSBs cause the accumulation of genomic rearrangements that promote tumorigenesis. For the repair of such DSBs, two distinct pathways are found to be in action that is: homologous recombination (HR) and nonhomologous end-joining (NHEJ) among which NHEJ pathway could be a chief DSB repair pathway in human somatic cells.[10] The repair of DSBs by NHEJ occurs in organisms ranging from bacteria to mammals, indicating that this type of repair has been conserved during evolution. This appears to reflect the major contribution of NHEJ to cell survival following exposure to agents that cause DSBs. In NHEJ pathway, Ku heterodimer which is composed of 69 and 83-kD polypeptides binds to the broken ends of DNA within seconds of their formation and then recruits the other downstream repair factors of the pathway, followed by the last step of rejoining through the recruitment of X-ray repair cross-complementing protein 4 (XRCC4) and DNA ligase IV to the DSBs.[11],[12],[13] XRCC4 interacts strongly with DNA ligase IV through a tandem BRCA1 carboxyl-terminal domain in a coiled-coil region, resulting in a formation of a highly stable complex.[14],[15] If, any mutation takes place in this complex will result in an imbalance of DSB repair activity.[16],[17] The final step of ligation in the NHEJ pathway of broken DNA ends is carried out by the (ATP) adenosine triphosphate dependent DNA ligase IV which is a ubiquitous phase.[18],[19],[20],[21] The DNA ligase IV is found important in the maintenance of genomic stability. Further, it is expected that cells defective in DSB repair will have more sensitivity towards chemotherapeutic agents. So, the study of DNA ligase has remained an attractive therapeutic target in cancers.[22],[23],[24] Based on this information, the current study aimed to evaluate the protein expression of core molecules of the NHEJ pathway that are Ku70/80, XRCC4, and DNA ligase IV and its effect on the disease outcome in patients with OSCC.


 > Materials and Methods Top


Patient selection and follow-up details

Formalin-fixed and paraffin-embedded (FFPE) tissue specimens from 100 patients with primary OSCC who underwent surgery were obtained from the histopathology department of the institute [Figure 1]. Informed consent was obtained from all individual participants included in the study. The detailed clinical data was obtained from the patients' case files maintained at the medical record department of the institute [Table 1]. For overall survival (OS) and relapse-free survival (RFS) analysis, patients were followed for a minimum period of 24 months or until death. Patients enrolled in survival analysis are depicted in [Figure 2]. This study has been approved by the institutional review committee for the dissertation/thesis/publication/conference presentation and the institutional ethics committee.
Figure 1: (Original) Schematic diagram of patients enrolled in the study

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Table 1: Clinicopathological characteristics of the oral squamous cell carcinoma patients

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Figure 2: (Original) Schematic diagram of patients enrolled in the study for survival analysis (a) patients enrolled in protein expression analysis and (b) patients enrolled in mRNA expression analysis

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RNA extraction and reverse transcription polymerase chain reaction

Out of a total of 100 patients enrolled in the present study, total RNA was extracted from a total of 66 snap-frozen OSCC tumor tissues using RNA iso-plus (Takara) solution. After measuring the yield of total extracted RNA, 1 μg of total RNA was reverse transcribed using iScript cDNA synthesis kit (Bio-Rad, California, US). The primer sequences for Ku70 were F: 5'-GATGCCTCCAAGGCTATGTTTGA-3', R: 5'-TTCTCGGTACCATAGAACACCACAG-3'.[25] Ku80 were F: 5'-ACGATTTGGTACAGATGGCACT-3', R: 5'-GCTCCTTGAAGACGCACAGTTT-3'[26] and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were F: 5'-CGGAGTCAACGGATTTGGTCGTAT-3', R: 5'-AGCCTTCTCCATGGTGGTGAAGAC-3'.[27] Following first-strand cDNA synthesis, an aliquot of cDNA was subjected to 40 cycles of PCR to determine the integrity of the GAPDH mRNA using a Q5 High-fidelity DNA polymerase kit (NEB, “Ipswich”, Massachusetts, USA). The amplified DNA fragment was 149 bp, 548 bp, and 309 bp for Ku70, Ku80, and GAPDH, respectively. Samples of the amplified products were then subjected to 2% agarose gel electrophoresis, stained with EtBr and visualized under gel documentation system (Alpha imager-EP, Santa Carla, CA, USA).

Immunohistochemistry and scoring

Immunohistochemistry (IHC) was carried out on FFPE OSCC tumor tissue sections with 4 μm-thicknesses to analyze the protein expression of Ku70/80, XRCC4 and DNA ligase IV. Sections were deparaffinized and rehydrated followed by blocking of endogenous peroxidase activity using 3% H2O2. Tissue antigenicity was retrieved using 10 mM tri-sodium citrate buffer. Nonspecific conjugations were blocked using a protein block provided in rabbit-specific HRP/DAB (ABC) detection IHC kit (Abcam, Cambridge, UK). Primary antibodies (Ku70/80, Genetex, 1:200; XRCC4, Santa Cruz, 1:200; DNA ligase IV, Genetex, 1:200) diluted in tris buffered saline were then applied to the sections and incubated overnight at 4°C. Antibody binding was visualized using the 3,3-diamino-benzidine as chromogen and counterstained with Mayer's hematoxylin, dehydrated in ethanol, mounted in dibutyl phthalate xylene, coverslipped, and observed under the light microscope.

The scoring method applied was a modified H-score method.[28] Data were divided into weak and strong expression groups by the median H-score values. Nuclear immune reactions were obtained for Ku70/80 and XRCC4, while for DNA ligase IV nuclear as well as cytoplasmic immune reactions were obtained.

Statistical analysis

Statistical analysis was performed using Statistical Package for Social Sciences version-20 (IBM, Armonk, New York, US). A two-tailed Chi-square test was used to examine the association between protein expression and clinicopathological parameters. Spearman's correlation coefficient (r) was used to calculate the correlation between the two parameters. The univariate survival was calculated using Kaplan–Meier survival function and tested for statistical significance using the log-rank statistics. For multivariate survival analysis, Cox forward stepwise regression model was used. Variables with or without prognostic potential in univariate survival analysis were included for multivariate survival analysis. P < 0.05 was taken into consideration to show statistical significance.


 > Results Top


mRNA expression of Ku70, Ku80, and association with clinicopathological parameters

Expression of Ku70 and Ku80 mRNA was examined in a total of 66 snap-frozen OSCC tumor tissues. The expression of Ku70 and 80 mRNA was detected in all the patients [Figure 3]. Expression of Ku70 and Ku80 mRNA, however, was not significantly correlated with any of the clinicopathological parameters such as age, gender, anatomic site, tobacco habit, tumor size, tumor stage, lymph node status, tumor differentiation and presence of lymphatic permeation and vascular permeation.
Figure 3: (Original) mRNA expression of Ku70, Ku80, and glyceraldehyde-3-phosphate dehydrogenase from the oral squamous cell carcinoma tumor samples from lanes 1-5 from left to right

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Protein expression of Ku70/80, X-ray repair cross-complementing protein 4, DNA ligase IV and association with clinicopathological parameters

IHC was performed to determine the protein expression of Ku70/80, XRCC4 and DNA ligase IV in a total of 100 patients with OSCC. For Ku70/80 and XRCC4, nuclear immunostaining was observed while for the DNA ligase IV, both nuclear as well as cytoplasmic immunostaining was observed [Figure 4]. For statistical evaluation, nuclear and cytoplasmic expressions were scored independently and compared separately. The positivity of Ku70/80, XRCC4 and DNA ligase IV was 99% (99/100), 100% (100/100), and 96% (96/100), respectively. Overall, expression of Ku70/80 and XRCC4 protein was higher as compared to DNA ligase IV protein expression in patients with OSCC [Figure 5]. Two-tailed Chi-square test revealed that Ku70/80 was significantly associated with patients without lymphatic permeation (P = 0.049), XRCC4 was significantly associated with tumor stage (P = 0.032) and locoregional aggressive disease (P = 0.010) while, nuclear DNA ligase IV was significantly associated with tobacco habituates (P = 0.010) [Table 2] and [Figure 6]. Intercorrelation of studied proteins has shown in [Table 3], indicating that XRCC4 protein expression was significantly positively correlated with Ku70/80 (P = 0.005) and DNA ligase IV (P = 0.005).
Figure 4: (Original) Representative immunohistochemistry image (original ×40) of (a) nuclear expression of Ku70/80, (b) nuclear expression of X-ray repair cross-complementing protein 4, and (c) nuclear and cytoplasmic expression of DNA ligase IV protein in oral squamous cell carcinoma tumor sections

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Figure 5: (Original) Protein expression analysis of Ku70/80, X-ray repair cross-complementing protein 4, nuclear DNA ligase IV and cytoplasmic DNA ligase IV

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Figure 6: (Original) (a) strong expression of Ku70/80 in lymph-node negative patients, (b) strong expression of X-ray repair cross-complementing protein 4 in stage III/IV patients, (c) strong expression of X-ray repair cross-complementing protein 4 in positive lymph node patients, and (d) strong expression of nuclear DNA ligase in tobacco habituate patients

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Table 2: Correlation of Ku70/80, XRCC4 and DNA ligase IV protein expression with clinicopathological parameters in oral squamous cell carcinoma patients

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Table 3: Intercorrelation of Ku70/80, XRCC4 and DNA ligase IV protein expression in oral squamous cell carcinoma patients

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Survival analysis

Using Kaplan–Meier univariate survival function, strong expression of Ku70 mRNA was found to be associated significantly with reduced OS (log-rank = 5.19, P = 0.023) while strong expression of XRCC4 protein was found as a significant prognosticator in developing disease relapse (log-rank = 5.744, P = 0.017) and reduced OS (log-rank = 4.291, P = 0.038) in patients with OSCC [Figure 7]. However, multivariate survival analysis of predictors using a multivariate Cox regression forward stepwise model revealed strong expression of XRCC4 as a significant independent prognosticator in predicting reduced OS (HR = 1.911, P = 0.047) [Table 4]. However, when multivariate survival analysis carried out including studied NHEJ markers along with clinicopathological parameters, weak expression of cytoplasmic expression of DNA ligase IV showed a trend towards significance as a prognosticator in predicting shorter OS (HR = 3.730, P = 0.053) in patients with OSCC [Table 4].
Figure 7: (Original) Survival analysis in oral squamous cell carcinoma patients (a) strong Ku70 mRNA expression associated with reduced overall survival, (b) strong X-ray repair cross-complementing protein 4 protein expression associated with reduced relapse-free survival, and (c) strong X-ray repair cross-complementing protein 4 protein expression associated with reduced overall survival

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Table 4: Multivariate overall survival analysis using Cox regression forward stepwise model in patients with OSCC

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 > Discussion Top


Although remarkable improvement has been made over the last decades in the understanding of the pathogenesis of OSCC, patients still die within a shorter period of time even after 1st-line treatment. Critical analysis of factors involved in survival and prognosis potentially leads to a better understanding of the pathogenesis of OSCC. Therefore, identification of patient and/or tumor-specific marker associated with increased or decreased survival time is an important stratagem. Although multiple studies have been conducted concerning the prognostic importance of different variables, the picture remains incomplete and unclear.

The current study demonstrated the significance of proteins having an important role in the repair of the DNA DSB and its impact on disease outcome in patients with OSCC. When studied NHEJ molecules were correlated with clinicopathological parameters, a strong expression of Ku70/80 protein was significantly associated with the absence of lymphatic permeation. In gastric carcinoma, the higher expression of Ku70/80 protein was correlated negatively with the clinical stage.[26] However, Alshareeda et al. in 2013 also found that Ku70/80 protein expression was associated with higher tumor grade (Grade III) and lymphovascular invasion in breast cancer indicative of its association with malignant behavior of breast cancer cells.[27] Such discrepancy in the results could be due to the different malignancy and phenotype. Further, the strong expression of XRCC4 protein was significantly associated with advanced-stage disease and lymph node involvement indicating its association with tumor aggressiveness. About DNA ligase IV protein, a significantly high incidence of strong nuclear DNA ligase IV protein expression was observed in tobacco habituates. While, a trend was observed in males patients with cancer of the buccal mucosa, demonstrating that the expression of DSB repair molecules has been elevated as a result of the increased oxidative stress due to frequent tobacco exposure to the buccal mucosa.

Univariate survival analysis revealed that the strong expression of Ku70 mRNA was significantly associated with a shorter OS. In accordance, in colorectal cancer, higher Ku70 mRNA expression was associated with poorer prognosis in rectal and advanced colorectal cancer.[29],[30] However, Pavón et al. in 2008 found that in patients with locally advanced head and neck squamous cell carcinoma (HNSCC), tumors with higher Ku70 mRNA expression had a significantly increased RFS and OS.[31] Discrepancy in the findings could be the outcome of the altered gene expression of Ku70 as a result of the several posttranscriptional modifications. However, in HNSCC, tumors expressing lower Ku80 mRNA had a reduced OS.[32] Further, a strong expression of XRCC4 protein was significantly associated with a high incidence of disease relapse and death in OSCC patients. In hypopharyngeal cancer patients with stage I to III disease, lower expression of the XRCC4 was tended to have better locoregional control.[33] The polymorphism in the XRCC4 gene is found to be susceptible to the development of various human cancers like urothelial bladder cancer, breast cancer, and pancreatic cancer.[34],[35],[36] Further, Datkhile et al. in 2016 found that the polymorphism in T allele of XRCC4 (G1394T) is responsible for higher genetic susceptibility to oral cancer and oral carcinogenesis which might be used as a novel useful marker for primary prevention and intervention.[37]

In the present study, cells expressing higher Ku70 mRNA expression found to have reduced OS. In agreement with this, in uterine cervical cancer biopsy sample and in rectal cancer, cells expressing low levels of Ku70 and Ku70/80 protein respectively, were radiosensitive and exhibited a significantly better RT outcome with improved survival.[29],[38] Further, silencing Ku70 by siRNA resulted in the downregulation of Ku70 which induces sensitization to etoposide, cisplatin or topotecan in the human cervical carcinoma HeLa cell line.[39] The lower expression of Ku70 or Ku80 in preradiation biopsy samples of the tumor was correlated with better objective responses, suggesting an association of low expression of Ku protein with high radiosensitivity in hypopharyngeal carcinoma.[33] In multivariate survival analysis including all established clinicopathological variables and studied DDR markers, cytoplasmic DNA ligase IV entered at step 3 in predicting shorter OS. While in multivariate survival analysis including studied NHEJ molecules revealed XRCC4 as an independent prognosticator in predicting shorter OS in patients with OSCC. In agreement with the present study, in human cells expressing defective DNA ligase IV, other DSBs repairing factors such as XRCC4 and XRCC4 like factor (XLF) failed to bind broken DNA molecules, indicating that participation of ligase IV is required for the substrate recognition by XRCC4 and XLF in vivo.[40] Further, in patients with nasopharyngeal cancer, Kim et al. reported a decrease in 5 years RFS and OS rate of patients with negative DNA ligase IV expression.[41]

Further, XRCC4 functions as a regulatory element and is required for precise end-joining of blunt DNA DSBs to stabilize DNA ligase IV, stimulate DNA ligase IV activity and directs DNA ligase IV to the site of the DNA breaks via its DNA binding capacity. Therefore, the association of strong XRCC4 and weak DNA ligase IV with reduced OS could be due to the polymorphism of XRCC4 or mutation in DNA ligase IV leading to the defective function in the repair of DNA DSBs. Although, broad analysis of certain experimental and clinical-epidemiologic results have suggested a role of receptor signaling such as estrogen receptor as a chief safeguard of genome stability and to have a strong interaction with DNA controlling and repairing pathways.[42] Hence, in the current study, further receptor expression analysis is warranted.


 > Conclusion Top


Collectively, in the present study, strong expression of Ku70 mRNA, XRCC4, and weak expression of cytoplasmic DNA ligase IV were significantly associated with reduced OS in OSCC patients indicating increased repair activity of DNA DSBs of the tumor cells by NHEJ pathway may lead to the progression of the disease. Further, the current study identified weak cytoplasmic DNA ligase IV as significant prognostic factors in predicting reduced RFS. While weak cytoplasmic DNA ligase IV and XRCC4 as a potential prognosticator in predicting reduced OS in patients with OSCC which might have great clinical implication in future for designing a treatment strategy.

Financial support and sponsorship

This study was financially supported by the Gujarat Cancer Society/The Gujarat Cancer and Research Institute.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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