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UHRF1 depletion sensitizes retinoblastoma cells to chemotherapeutic drugs via downregulation of XRCC4.

ABSTRACT: UHRF1 (ubiquitin-like with PHD and ring finger domains 1) is highly expressed in various human cancers including retinoblastoma, and associated with tumor-promoting effects such as inhibition of apoptosis and high proliferation. However, the molecular mechanisms underlying tumor-promoting functions of UHRF1 in retinoblastoma still remain elusive. Here, we show that stable knockdown of UHRF1 renders retinoblastoma cells sensitized to conventional chemotherapeutic drugs such as etoposide and camptothecin, resulting in enhanced DNA damage and apoptotic cell death. We found that UHRF1-depleted retinoblastoma cells can recognize DNA damages normally but have markedly low expression of XRCC4 (X-ray repair cross complementing 4) among the components of nonhomologous end-joining (NHEJ) repair complex. Conversely, overexpression of UHRF1 increased the XRCC4 expression and stable knockdown of XRCC4 sensitized retinoblastoma cells to etoposide treatment, suggesting that XRCC4 is a key mediator for the drug sensitivity upon UHRF1 depletion in retinoblastoma cells. Consistent with the findings, chromatin association of DNA ligase IV in response to acute DNA damage was found to be significantly reduced in UHRF1-depleted retinoblastoma cells and functional complementation for XRCC4 in UHRF1-depleted cells attenuated the drug sensitivity, demonstrating that XRCC4 downregulation in UHRF1-depleted cells impaired DNA repair and consequently induced robust apoptosis upon genotoxic drug treatment. In human primary retinoblastoma, high expression of UHRF1 and XRCC4 could be detected, and elevated XRCC4 expression correlated with reduced apoptosis markers, implying that UHRF1-mediated XRCC4 upregulation under pathophysiological conditions triggered by RB1 gene inactivation may confer protection against endogenous DNA damages that arise during retinoblastoma development. Taken together, these results present a new mechanistic insight into how UHRF1 mediates its tumor-promoting functions in retinoblastoma, and also provide a basis for UHRF1 targeting to improve the efficacy of current chemotherapy for retinoblastoma treatment.

SUBMITTER: He H

PROVIDER: S-EPMC5833858 | biostudies-other | 2018 Feb

REPOSITORIES: biostudies-other

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UHRF1 depletion sensitizes retinoblastoma cells to chemotherapeutic drugs via downregulation of XRCC4.

He Heng H Lee Chunsik C Kim Jong Kyong JK

Cell death & disease 20180207 2

UHRF1 (ubiquitin-like with PHD and ring finger domains 1) is highly expressed in various human cancers including retinoblastoma, and associated with tumor-promoting effects such as inhibition of apoptosis and high proliferation. However, the molecular mechanisms underlying tumor-promoting functions of UHRF1 in retinoblastoma still remain elusive. Here, we show that stable knockdown of UHRF1 renders retinoblastoma cells sensitized to conventional chemotherapeutic drugs such as etoposide and campt ...[more]

PMID: 29415984

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Project description:Anoikis is a type of apoptosis induced by cell detachment from the extracellular matrix (ECM), which removes mislocalized cells. Acquisition of anoikis resistance is critical for cancer cells to survive during circulation and, thus, metastasize at a secondary site. Although the sensitization of cancer cells to anoikis is a potential strategy to prevent metastasis, the mechanism underlying anoikis resistance is not well defined. Although family with sequence similarity 188 member B (FAM188B) is predicted as a new deubiquitinase (DUB) member, its biological function has not been fully studied. In this study, we demonstrated that FAM188B knockdown sensitized anoikis of lung cancer cell lines expressing WT-EGFR (A549 and H1299) or TKI-resistant EGFR mutant T790M/L858R (H1975). FAM188B knockdown using si-FAM188B inhibited the growth of all three human lung cancer cell lines cultured in both attachment and suspension conditions. FAM188B knockdown resulted in EGFR downregulation and thus decreased its activity. FAM188B knockdown decreased the activities of several oncogenic proteins downstream of EGFR that are involved in anoikis resistance, including pAkt, pSrc, and pSTAT3, with little changes to their protein levels. Intriguingly, si-FAM188B treatment increased EGFR mRNA levels but decreased its protein levels, which was reversed by treatment with the proteasomal inhibitor MG132, indicating that FAM188B regulates EGFR levels via the proteasomal pathway. In addition, cells transfected with si-FAM188B had decreased expression of FOXM1, an oncogenic transcription factor involved in cell growth and survival. Moreover, FAM188B downregulation reduced metastatic characteristics, such as cell adhesion, invasion, and migration, as well as growth in 3D culture conditions. Finally, tail vein injection of si-FAM188B-treated A549 cells resulted in a decrease in lung metastasis and an increase in mice survival in vivo. Taken together, these findings indicate that FAM188B plays an important role in anoikis resistance and metastatic characteristics by maintaining the levels of various oncogenic proteins and/or their activity, leading to tumor malignancy. Our study suggests FAM188B as a potential target for controlling tumor malignancy.

Project description:BackgroundChanges in chromosome number or structure as well as supernumerary centrosomes and multipolar mitoses are commonly observed in human tumors. Thus, centrosome amplification and mitotic checkpoint dysfunctions are believed possible causes of chromosomal instability. The Retinoblastoma tumor suppressor (RB) participates in the regulation of synchrony between DNA synthesis and centrosome duplication and it is involved in transcription regulation of some mitotic genes. Primary human fibroblasts were transfected transiently with short interfering RNA (siRNA) specific for human pRb to investigate the effects of pRb acute loss on chromosomal stability.ResultsAcutely pRb-depleted fibroblasts showed altered expression of genes necessary for cell cycle progression, centrosome homeostasis, kinetochore and mitotic checkpoint proteins. Despite altered expression of genes involved in the Spindle Assembly Checkpoint (SAC) the checkpoint seemed to function properly in pRb-depleted fibroblasts. In particular AURORA-A and PLK1 overexpression suggested that these two genes might have a role in the observed genomic instability. However, when they were post-transcriptionally silenced in pRb-depleted fibroblasts we did not observe reduction in the number of aneuploid cells. This finding suggests that overexpression of these two genes did not contribute to genomic instability triggered by RB acute loss although it affected cell proliferation. Acutely pRb-depleted human fibroblasts showed the presence of micronuclei containing whole chromosomes besides the presence of supernumerary centrosomes and aneuploidy.ConclusionHere we show for the first time that RB acute loss triggers centrosome amplification and aneuploidy in human primary fibroblasts. Altogether, our results suggest that pRb-depleted primary human fibroblasts possess an intact spindle checkpoint and that micronuclei, likely caused by mis-attached kinetochores that in turn trigger chromosome segregation errors, are responsible for aneuploidy in primary human fibroblasts where pRb is acutely depleted.

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UHRF1 depletion sensitizes retinoblastoma cells to chemotherapeutic drugs via downregulation of XRCC4.

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UHRF1 depletion sensitizes retinoblastoma cells to chemotherapeutic drugs via downregulation of XRCC4.

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