Novel role for non-homologous end joining in the formation of double minutes in methotrexate-resistant colon cancer cells

Abstract

BackgroundGene amplification is a frequent manifestation of genomic instability that plays a role in tumour progression and development of drug resistance. It is manifested cytogenetically as extrachromosomal double minutes (DMs) or intrachromosomal homogeneously staining regions (HSRs). To better understand the molecular mechanism by which HSRs and DMs are formed and how they relate to the development of methotrexate (MTX) resistance, we used two model systems of MTX-resistant HT-29 colon cancer cell lines harbouring amplified DHFR primarily in (i) HSRs and (ii) DMs.ResultsIn DM-containing cells, we found increased expression of non-homologous end joining (NHEJ) proteins. Depletion or inhibition of DNA-PKcs, a key NHEJ protein, caused decreased DHFR amplification, disappearance of DMs, increased formation of micronuclei or nuclear buds, which correlated with the elimination of DHFR, and increased sensitivity to MTX. These findings indicate for the first time that NHEJ plays a specific role in DM formation, and that increased MTX sensitivity of DM-containing cells depleted of DNA-PKcs results from DHFR elimination. Conversely, in HSR-containing cells, we found no significant change in the expression of NHEJ proteins. Depletion of DNA-PKcs had no effect on DHFR amplification and resulted in only a modest increase in sensitivity to MTX. Interestingly, both DM-containing and HSR-containing cells exhibited decreased proliferation upon DNA-PKcs depletion.ConclusionsWe demonstrate a novel specific role for NHEJ in the formation of DMs, but not HSRs, in MTX-resistant cells, and that NHEJ may be targeted for the treatment of MTX-resistant colon cancer.

Inhibiting homologous recombination decreases extrachromosomal amplification but has no effect on intrachromosomal amplification in methotrexate‐resistant colon cancer cells

“…Thus, we hypothesize that the formation of HSR and DMs may depend on different molecular mechanisms. For example, NHEJ is mainly involved in DM formation but not HSR . In our study, we observed different roles of HR in the formation of DM and HSR.…”

“…Thus, DSB repair pathways may be involved in gene amplification. In our previous study, we demonstrated a special role for one of the DSB pathway, NHEJ, in the formation of DMs . Among DSB repair pathways, homologous recombination (HR) is a classical mechanism that consists of multiple interrelated pathways.…”

“…Micronuclei (MNs) and nuclear buds (NBUDs) are the main pathways for export of nuclear material, including amplified DNA, DNA repair complexes and excess chromosomes . In previous studies, the application of DNA synthesis or repair inhibitors, such as hydroxyurea, gemcitabine and NU7026, has been found to eliminate gene amplification by forming MN/NBUDs . The formation of MN/NBUDs tightly depends on the cell cycle, and the HR core protein, BRCA1, is a regulator of cell cycle checkpoints .…”

Cell-Free eccDNAs: A New Type of Nucleic Acid Component for Liquid Biopsy?

“…In pathological conditions, eccDNAs are found to play roles in human diseases and disease treatments, especially for larger eccDNAs (episomes and DMs) with their functions being better characterized. For example, DMs, exclusively found in tumor cells (13) and drug-resistance cells (43), significantly elevate the active gene transcripts to promote tumor progression. Because of their self-replication and uneven segregation, DMs are related to tumor cell heterogeneity and adaptation (44).…”

MiR-505 mediates methotrexate resistance in colorectal cancer by targeting RASSF8

“…revealed that MTX might inhibit breast cancer cell growth through G2/M elongation . It was also reported that MTX, coupled with 5‐fluorouracil, can impact the cell proliferation of cancer, including colon cancer . Nonetheless, the in‐depth mechanism of MTX in colorectal cancer remains unknown.…”