Lack of associations between LIG3 gene polymorphisms and neuroblastoma susceptibility in Chinese children (2024)

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Supplementary Materials

Abstract

Accumulating evidence suggests that dysregulation of the DNA non-hom*ologous end-joining (NHEJ) repair system is a causative factor in many cancers, including high-risk neuroblastoma. A number of studies have shown that polymorphisms in the DNA ligase III (LIG3) gene, one of the key genes in the error-prone alternative NHEJ (a-NHEJ) pathway for DNA double-strand break (DSB) repair, are associated with a variety of cancers. Nevertheless, whether LIG3 polymorphisms contribute to neuroblastoma risk remains unknown. We investigated the correlation between neuroblastoma susceptibility and two LIG3 polymorphisms (rs1052536 C>T and rs4796030 A>C) among 469 neuroblastoma patients and 998 healthy controls from China. Our results failed to detect any relationship between the analyzed SNPs and neuroblastoma risk in either overall analysis or stratification analysis. These results suggest that rs1052536 C>T and rs4796030 A>C are unrelated to neuroblastoma susceptibility in the Chinese population. Further studies with larger sample sizes and multiple ethnicities are necessary to verify our results.

Keywords: neuroblastoma, LIG3, polymorphism, case-control, susceptibility

Introduction

Neuroblastoma is one of the most common extracranial solid tumors in childhood. These tumors originate from abnormally differentiated or undifferentiated sympathetic nervous system stem cells and demonstrate highly heterogeneous biological characteristics and clinical manifestations. A minority of cases can resolve themselves, but most exhibit distant metastases at the time of diagnosis. Along with the advancement of treatment technology, the 5-year survival rate of low/medium-risk neuroblastoma has increased to more than 90%, but the 5-year survival rate of children with high-risk neuroblastoma is still less than 50% ¹. Notably, approximately 60% of patients are diagnosed with high-risk neuroblastoma.

The etiology of neuroblastoma is currently unclear, but accumulating evidence implicates disorders of DNA damage repair in abnormal cell proliferation ². In neuroblastoma, genome amplification of the MYCN gene encoding N-myc proto-oncogene protein (N-Myc) and segmental chromosomal alterations, including 1p heterozygous deletions, 11q heterozygous deletions, and 17q increases, are associated with tumor progression and poor prognosis ³. Pashaiefar et al. ⁴ found that expression of the DNA ligase III (LIG3) gene was abnormally elevated in bone marrow specimens from patients with acute myeloid leukemia. Furthermore, MYCN binds to the promoter region of LIG3, thereby increasing its transcriptional activity and leading to enhanced cellular tyrosine kinase activity ⁴. Newman et al. ⁵ found that DNA double-strand breakage contributes to human neuroblastoma and that the alternative (a)-NHEJ pathway involving LIG3 is a therapeutic target for high-risk neuroblastoma. LIG3 polymorphisms influencing expression may contribute to neuroblastoma susceptibility. In fact, Landi et al. ⁶ found that the LIG3 rs1052536 single nucleotide polymorphism (SNP) was significantly associated with lung cancer risk among the young population. Moreover, genetic polymorphisms of LIG3 are strongly related to breast cancer prognosis ⁷. However, whether LIG3 polymorphisms are also associated with neuroblastoma risk remains unclear. Therefore, we conducted a three-center case-control study including 469 neuroblastoma patients and 998 controls to investigate the association between LIG3 gene polymorphisms and neuroblastoma susceptibility in Chinese children.

Materials and Methods

Results

Discussion

DNA double-strand breaks (DSBs) are the most severe form of damage to the integrity and stability of the genome. Failure to repair this damage or errors in repair can lead to genome instability and abnormalities in cell proliferation. The main DSB repair mechanisms in mammalian cells include the hom*ologous recombination (HR) repair and non-hom*ologous end-joining (NHEJ) repair pathways. NHEJ is divided into two pathways: classical NHEJ (c-NHEJ) and a-NHEJ ¹⁰. There is accumulating evidence that NHEJ dysregulation is a causative factor in many cancers, such as neuroblastoma ⁵, breast cancer ⁷, chronic myeloid leukemia ¹¹, lung cancer ⁶, esophageal squamous cell carcinoma ¹², pancreatic cancer ¹³, and non-oral or squamous cell carcinoma ¹⁴. The a-NHEJ pathway participates in binding, cleavage, and ligation of DNA breaks and depends on PARP1, the MRN complex, LIG1, and LIG3, excluding several key genes of the c-NHEJ pathway. Compared to c-NHEJ, a-NHEJ is more prone to false repairs, with missing or unbalanced translocations. Chromosomal translocation is a driving factor for tumorigenesis in a variety of cell types, and the error-prone repair characteristics of a-NHEJ make it a key factor for genomic instability and tumorigenesis ¹⁵.

The human LIG3 gene is located on chromosome 17 q11.2-q12 ¹⁶. Unlike LIG1 and LIG4, LIG3 encodes three or four different DNA ligase polypeptides (including DNA ligases IIIα and IIIβ) with distinct functions in amphibians and mammals ¹⁷. Among them, nuclear Lig IIIα is mainly involved in excision repair and a-NHEJ repair ¹⁸, and mitochondrial Lig IIIα is involved in mitochondrial genome replication and repair ¹⁹. Furthermore, Lig IIIα is frequently overexpressed in cancer cells and is a biomarker for increased dependence upon a-NHEJ for DSB repair ¹⁷. It is also considered a potential novel target for tumor therapy ²⁰.

A growing body of research indicates that LIG3 SNPs are significantly associated with cancer susceptibility and recurrence ^{5-7, 11-14}. Landi et al. ⁶ found that LIG3 rs1052536 is significantly associated with susceptibility to lung cancer among young people. Surprisingly, breast cancer patients with LIG3 rs1052536 tend to demonstrate longer progression-free survival ⁷. Zhu et al. ¹⁴ reported that LIG3 rs4796030 can significantly modify the recurrence risk of non-oropharyngeal squamous cell carcinoma. Other LIG3 SNPs are also significantly associated with susceptibility to esophageal squamous cell carcinoma and pancreatic cancer ^{12, 13}. Daniele et al. ²¹ reported that multiple myeloma patients with elevated expression of LIG3 mRNA tend to have a worse prognosis, and LIG3 mRNA expression was elevated during disease progression and relapse. They also found that LIG3 knockdown significantly increased DNA damage and ultimately inhibited the growth of multiple myeloma cells in vivo and vitro. In the current study, we comprehensively assessed the associations between LIG3 polymorphisms and neuroblastoma risk but did not detect a significant association. Erika et al. ⁵ found that the expression levels of a-NHEJ proteins, such as LIG3, LIG1, and PARP1, were significantly upregulated in high-risk neuroblastoma cells compared to those in normal cells. Reduction in LIG3 and LIG1 expression levels in neuroblastoma cells led to DSB accumulation and cell death. In addition, they also found that patients with high expression of LIG3 in neuroblastoma cells had poorer prognoses. Thus, LIG3 may have a tumor-promoting role in neuroblastoma. Since LIG3 SNPs can affect the expression and activity of LIG3, we speculate that neuroblastoma patients with LIG3 SNPs may have longer overall survival and (or) tumor-free survival than those with higher expression of LIG3 mRNA or without these SNPs, although this speculation needs confirmation. If this hypothesis can be further validated, the use of mutant genes to induce LIG3 loss-of-function may be useful for the treatment of high-risk neuroblastoma patients.

This is the first study to assess the associations between LIG3 SNPs (rs1052536 and rs4796030) and susceptibility to neuroblastoma in Chinese children; however, there are several limitations. First, this study is retrospective and may be limited by information and selection bias. Neuroblastoma is a heterogeneous tumor with a complex etiology, and we failed to obtain information on potentially important environmental factors, such as living environment, diet, and pregnancy exposure in this study. These factors should be addressed in the future. Second, despite the relatively large total sample sizes included in the study, some subgroup sample sizes were relatively small, which inevitably diminished the statistical power. Third, only two polymorphisms in the LIG3 gene were studied. Other potentially functional LIG3 polymorphisms may also modify the encoded helicase or the activity of gene and thus should be involved in further study. Fourth, whether LIG3 SNPs are closely related to prognosis of neuroblastoma was not studied in this study, which needs to be studied further. Finally, because the subjects were mainly from the Han population in different parts of China, the conclusions drawn may not be directly applicable to people of other ethnicities.

In conclusion, these results do not support a significant association between LIG3 gene polymorphisms and susceptibility to neuroblastoma; it is necessary to expand the sample size and conduct epidemiological studies on additional polymorphic loci in different ethnic groups.

Supplementary Material

Acknowledgments

This work was supported by grants from the Youth Science and Technology New Star Project of Shaanxi Province (No: 2018KJXX-050), Basic Scientific Research fee of Xi'an Jiaotong University (No: XJJ2018126 and No: YJ(ZD)201704).

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