Identification of genes associated with methotrexate resistance in methotrexate-resistant osteosarcoma cell lines

Yang, Xiaorong; Xiong, Yan; Duan, Hong; Gong, Renmin

doi:10.1186/s13018-015-0275-8

Cited by 26 publications

(23 citation statements)

References 36 publications

(38 reference statements)

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“…As previously reported, the down-regulated DEGs of MTX-resistant osteosarcoma cell lines were enriched in the mitotic cell cycle, cell cycle, and DNA replication pathways, which were also down-regulated in our MTX-resistant colon cancer cells. This result might explain the role of MTX in inhibiting dihyrofolate reductase ( DHFR ) and preventing tumor cells from proliferating in both cases 38, 39 . In addition, down-regulated expression of MMR genes, which affects G2/M cell cycle arrest and apoptosis, might prevent proper checkpoint and cell-death signaling; therefore, this phenomenon could contribute to the malfunctioning of DNA replication 40 .…”

Section: Resultsmentioning

confidence: 98%

Genomic and transcriptomic analyses reveal a tandem amplification unit of 11 genes and mutations of mismatch repair genes in methotrexate-resistant HT-29 cells

Kim

Shin

Seo

2020

Preprint

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DHFR gene amplification is present in methotrexate (MTX)-resistant colon cancer cells and acute lymphoblastic leukemia. However, little is known about DHFR gene amplification due to difficulties in quantifying amplification size and recognizing the repetitive rearrangements involved in the process. In this study, we have proposed an integrative framework to characterize the amplified region by using a combination of single-molecule real time sequencing, next-generation optical mapping, and chromosome conformation capture (Hi-C). Amplification of the DHFR gene was optimized to generate homogenously amplified patterns. The amplification units of 11 genes, from the DHFR gene to the ATP6AP1L gene position on chromosome 5 (~2.2Mbp), and a twenty-fold tandemly amplified region were verified using longrange genome and RNA sequencing data. In doing so, a novel inversion at the start and end positions of the amplified region as well as frameshift insertions in most of the MSH and MLH genes were detected. These might stimulate chromosomal breakage and cause the dysregulation of mismatch repair pathways. Using Hi-C technology, high adjusted interaction frequencies were detected on the amplified unit and unsuspected position on 5q, which could have a complex network of spatial contacts to harbor gene amplification. Characterizing the tandem gene-amplified unit and genomic variants as well as chromosomal interactions on intra-chromosome 5 can be critical in identifying the mechanisms behind genomic rearrangements. These findings may give new insight into the mechanisms underlying the amplification process and evolution of drug resistance. Results Determination of MTX-resistant HT-29 cellsWhile generating MTX-resistant HT-29 cells (selected clone: C1-2) from singe-cell selection and MTX sensitization as previously described 15 , dramatic morphological changes in the cells themselves were detected in the form of rounded and circular shapes at the 1 st cycle of sensitization as well as rod and irregular shapes at the 2 nd cycle (Supplementary Fig. 1). The original shapes were again observed at the 3 rd cycle of sensitization, which might indicate that the HT-29 cells were becoming resistant to MTX and rapidly grew up under high MTX concentration.As expected, DHFR expression, which was normalized by the B2M housekeeping gene, was steadily increased from the 1 st to 3 rd cycle, as the HT-29 clones became resistant to MTX (Supplementary Fig. 2a). After confirming these morphological changes and increased DHFR expression, the DHFR copy number was measured in several clones.The clones' cycle quantification values (Ct) were used to compute the rate of copy number. These values dropped from 26.04 to 19.99, as the number of cycles increased ( Supplementary Table 1). Interestingly, there was a dramatic increase in the DHFR copy number between the 1 st (0.97 copies) and 2 nd (54.83 copies) cycles ( Supplementary Fig. 2b).Based on these results, we ascertained that a specific time period and set of conditions were required for clones to su...

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Section: Resultsmentioning

confidence: 98%

Genomic and transcriptomic analyses reveal a tandem amplification unit of 11 genes and mutations of mismatch repair genes in methotrexate-resistant HT-29 cells

Kim

Shin

Seo

2020

Preprint

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“…Transactivation and upregulation of FPGS have been reported to promote the cytotoxicity of MTX in many cancer cell lines and vice versa. Treatment with histone deacetylase inhibitors (HDA-Cis) resulted in increased FPGS expression and higher intracellular accumulation of long-chain MTX-PG [3][4][5][6][7] in the relevant all models through epigenetic mechanisms involving histone modifications and chromatin remodelling and NFY-B-and Sp1-mediated recruitment of HDAC1 to the FPGS promoter [41]. Some fusion proteins, such as TEL-AML1 and E2A-PBX1, suppress FPGS transcription by recruiting co-repressors (mSin3A, Rb) and HDAC1 to the FPGS promoter region [42].…”

Section: Discussionmentioning

confidence: 99%

“…To date, the mechanisms underlying MTX‐resistance remain largely unknown. Several mechanistic investigations have been carried out based on in vitro systems using drug‐induced MTX‐resistant OS cell lines, which are cell lines with acquired MDR and thus do not faithfully reflect mechanisms underlying de novo MTX‐resistance in OS patients .…”

Section: Introductionmentioning

confidence: 99%

SKA1 induces de novo MTX‐resistance in osteosarcoma through inhibiting FPGS transcription

Min²,

Lin

et al. 2019

The FEBS Journal

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De novo methotrexate (MTX)‐resistance, whose underlying mechanism remains largely unknown, usually leads to very poor prognosis in patients with osteosarcoma (OS). In this study, we established the de novo MTX‐resistant OS cell line SF‐86 and identified the candidate gene spindle and kinetochore associated complex subunit 1 (SKA1) as potentially related to de novo MTX‐resistance. Analysis of a cohort of 95 OS patients demonstrated that SKA1 overexpression significantly correlated with de novo MTX‐resistance and poor 5‐year survival. Mechanistically, SKA1 overexpression lead to a downregulation of folylpoly‐γ‐glutamate synthetase (FPGS), a key enzyme that converts MTX into its active form, MTX‐PG. We further demonstrated that SKA1 interacts with DNA‐directed RNA polymerase II subunit RPB3. ChIP analysis revealed that RPB3 binds the promoter region of the FPGS gene and triggers FPGS transcription upon MTX treatment in SW1353, a MTX‐sensitive OS cell line lacking endogenous SKA1 expression. On the contrary, this process is blocked in SF‐86 cells due to the formation of an inhibitory SKA1‐RPB3 complex. Furthermore, downregulation of SKA1 levels restores MTX sensitivity in SF‐86. Collectively, our study has established the de novo MTX‐resistant cell line SF‐86 and identified SKA1 as a novel regulator of FPGS, playing a key role in the development of de novo MTX‐resistance in OS.

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“…In this study, YM-155 mediated a loss of many of these processes including CCNB1 [-2.5, p<0.001] and CCNB2 [-2.77, p<0.001] (which encodes G2/mitotic-specific cyclin-B 1 and 2 proteins) cyclin B/CDK1, BORA, AURKB, CENPA, CCNE2 and its target PLK1 [-2.41, p<0.001], all of which are essential for mitotic recovery after DNA damage (80)(81)(82)(83)(84)(85). Many of these cycle checkpoint transcripts are reportedly overexpressed in aggressive chemo resistant tumors (86)(87)(88)(89)(90)(91) leading to unbridled proliferation (92), poor prognosis (93) to which small-molecule inhibitors that target these kinases are currently being tested as anticancer drugs (94)(95)(96). Kinase inhibition would indirectly prevent Plk1 activation, initiate G2 arrest (97), trigger a simultaneous rise in tumor suppressors-p53-inducible p21 tumor, Wee1, Myt1, p21 (98), as well as proteasomal Bcl-2 degradation (99) all contributing to cytostatic mediated apoptosis (100)(101)(102)(103)(104)(105)(106)(107).…”

Section: Discussionmentioning

confidence: 99%

Effects of Sepantronium Bromide (YM-155) on the Whole Transcriptome of MDA-MB-231 Cells: Highlight on Impaired ATR/ATM Fanconi Anemia DNA Damage Response

Mazzio

Lewis

Elhag

et al. 2018

Cancer Genomics Proteomics

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Sepantronium bromide (YM-155) is believed to elicit apoptosis and mitotic arrest in tumor cells by reducing (BIRC5, survivin) mRNA. In this study, we monitored changes in survivin mRNA and protein after treating MDA-MB-231 cells with YM-155 concurrent with evaluation of whole transcriptomic (WT) mRNA and long intergenic non-coding RNA at 2 time points: 8 h sub-lethal (83 ng/mL) and 20 h at the LC (14.6 ng/mL). The data show a tight association between cell death and the precipitating loss of survivin protein and mRNA (-2.67 fold-change (FC), p<0.001) at 20 h, questioning if the decline in survivin is attributed to cell death or drug impact. The meager loss of survivin mRNA was overshadowed by enormous differential change to the WT in both magnitude and significance for over 2000 differentially up/down-regulated transcripts: (+22 FC to -12 FC, p<0.001). The data show YM-155 to up-regulate transcripts in control of circadian rhythm (NOCT, PER, BHLHe40, NFIL3), tumor suppression (SIK1, FOSB), histone methylation (KDM6B) and negative feedback of NF-kappa B signaling (TNFAIP3). Down-regulated transcripts by YM-155 include glucuronidase (GUSBP3), numerous micro-RNAs, DNA damage repair elements (CENPI, POLQ, RAD54B) and the most affected system was the ataxia-telangiectasia mutated (ATM)/Fanconi anemia E3 monoubiquitin ligase core complexes (FANC transcripts - A/B/E/F/G/M), FANC2, FANCI, BRCA1, BRCA2, RAD51, PALB2 gene and ATR (ATM- and Rad3-Related) pathway. In conclusion, these findings suggest that a primary target of YM-155 is the loss of replicative DNA repair systems.

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Identification of genes associated with methotrexate resistance in methotrexate-resistant osteosarcoma cell lines

Cited by 26 publications

References 36 publications

Genomic and transcriptomic analyses reveal a tandem amplification unit of 11 genes and mutations of mismatch repair genes in methotrexate-resistant HT-29 cells

Genomic and transcriptomic analyses reveal a tandem amplification unit of 11 genes and mutations of mismatch repair genes in methotrexate-resistant HT-29 cells

SKA1 induces de novo MTX‐resistance in osteosarcoma through inhibiting FPGS transcription

Effects of Sepantronium Bromide (YM-155) on the Whole Transcriptome of MDA-MB-231 Cells: Highlight on Impaired ATR/ATM Fanconi Anemia DNA Damage Response

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