Emerging insights into alternative end‑joining: Mechanisms, genome instability and therapeutic opportunities in cancer (Review)

Abstract

Genome instability is a central hallmark of cancer, driven by aberrant DNA damage responses that facilitate tumor evolution and resistance to therapy. Although canonical non‑homologous end joining and homologous recombination are well‑characterized pathways for repairing DNA double‑strand breaks (DSBs), recent advances have revealed that cancer cells increasingly depend on alternative end‑joining (alt‑EJ) to survive persistent DNA damage that arises from intrinsic stresses or external therapies. Alt‑EJ, characterized by its reliance on microhomologous sequences at DSB sites, promotes mutation accumulation and chromosomal rearrangements, thereby driving genomic instability and tumor progression. Despite its pivotal role in cancer biology, the molecular regulation, contextual determinants and dualistic role of alt‑EJ in maintaining genome integrity compared with promoting instability remain incompletely understood. The present review integrated the latest mechanistic insights into alt‑EJ, elucidated its regulatory networks and interactions with canonical DSB repair pathways and discussed its consequences for cancer genome integrity and evolution. Furthermore, it highlighted the emerging potential of alt‑EJ as a therapeutic vulnerability for cancer, underscoring the urgent need to translate these discoveries into innovative treatment strategies aimed at overcoming therapy resistance and improving patient outcomes.

Keywords: DNA damage response; DNA double strand break repair; alternative end‑joining; cancer therapeutic targets; genome instability.

Figure 1

Major pathways for DSB repair. NHEJ begins with Ku70-Ku80 hetero-dimer attaching to DNA ends. DNA-PKcs recruitment and auto-phosphorylation bring the DNA ends together and allow ligation by XRCC4-LIG4 and XLF or PAXX. Resection by MRN complex and CtIP promotes homology-directed repair. Long-range resection creates RPA-coated ssDNA overhangs using BLM-DNA2 helicase-nuclease or EXO1 nucleases. HR occurs when BRCA1, PALB2 and BRCA2 facilitate loading RAD51 onto ssDNA and displace RPA. RAD51 nucleoprotein filaments invade the DNA-synthesis template sister chromatids. Alternatively, substantial resection generates a substrate for SSA, where RAD52 promotes homologous sequence annealing on each DNA end. ERCC1 and XPF handle 3'single-stranded flaps for LIG1-mediated DNA ligation. DSB resection also activates alt-EJ via PARP1, where POLQ anneals short homologous sequences, synthesizes DNA and re-ligates DNA ends using LIG1 or LIG3. DSB, DNA double-strand break; NHEJ, non-homologous end joining; DNA-PKcs, DNA-dependent protein kinase catalytic subunit; ssDNA, single-stranded DNA; HR, homologous recombination; alt-EJ, alternative end-joining; PARP, poly (ADP-ribose) polymerase; XRCC4, X-ray repair cross-complementing protein 4; LIG4, DNA ligase 4; XLF, XRCC4-like factor; PAXX, paralogue of XRCC4 and XLF; CtIP, c-terminal-binding protein-interacting protein; RPA, replication protein A; BLM, Bloom syndrome helicase; DNA2, DNA replication helicase/nuclease 2; EXO1, exonuclease 1; BRCA1/2, breast cancer gene 1/2; PALB2, partner and localizer of BRCA2; SSA, single strand annealing; ERCC1, excision repair cross complementation group 1; XPF, xeroderma pigmentosum group F; POLQ, DNA polymerase theta; LIG1/3, DNA ligase1/3; Ins/del, insertion or deletion; LOH, loss of heterogeneity; nt, nucleotide.

Figure 2

Schematic illustration of alt-EJ effectors. The alt-EJ pathway is influenced by several factors. TGFβ signaling begins in the cytoplasm and activates Smad proteins and non-canonical pathways to regulate DDR through miR-182 and other mechanisms (24,25,78). Loss of TGFβ signaling, such as with the effects induced by HPV, causes enhanced alt-EJ activity (25,79). The HPV oncoprotein E7 directs DNA repair to alt-EJ (83). PLK1 facilitate RHINO to accumulate in the M phase recruit POLQ to the break site (69). APE2 interacts with POLQ in an epistatic way for alt-EJ (63). CtIP, Mre11 and BLM proteins regulate DNA end resection, which subsequently facilitates the process of strand annealing and the coupling of fragmented DNA ends through the annealing of microhomologies. alt-EJ, alternative end-joining; DDR, DNA damage response; TGFβ, transforming growth factor-beta; Smad, suppressor of mothers against decapentaplegic; miR, microRNA; HPV, human papillomavirus; PLK1, polo-like kinase 1; APE2, apurinic/apyrimidinic endodeoxyribonuclease 2; RHINO, Rad9-Hus1-Rad1-interacting nuclear orphan; POLQ, DNA polymerase θ; CtIP, c-terminal-binding protein-interacting protein; MRE11, Meiotic recombination 11; BLM, Bloom syndrome helicase; PARP, poly (ADP-ribose) polymerase.