SGO1 is involved in the DNA damage response in MYCN-amplified neuroblastoma cells

Abstract

Shugoshin 1 (SGO1) is required for accurate chromosome segregation during mitosis and meiosis; however, its other functions, especially at interphase, are not clearly understood. Here, we found that downregulation of SGO1 caused a synergistic phenotype in cells overexpressing MYCN. Downregulation of SGO1 impaired proliferation and induced DNA damage followed by a senescence-like phenotype only in MYCN-overexpressing neuroblastoma cells. In these cells, SGO1 knockdown induced DNA damage, even during interphase, and this effect was independent of cohesin. Furthermore, MYCN-promoted SGO1 transcription and SGO1 expression tended to be higher in MYCN- or MYC-overexpressing cancers. Together, these findings indicate that SGO1 plays a role in the DNA damage response in interphase. Therefore, we propose that SGO1 represents a potential molecular target for treatment of MYCN-amplified neuroblastoma.

Figure 1. Sgo1 expression increases with neuroblastoma progression, and SGO1 expression is elevated in MYCN-amplified neuroblastoma cell lines.

(a) Quantitative RT-PCR analyses of Sgo1 mRNA levels in precancerous lesions from four hemizygous MYCN-Tg mice (1.6 or 2 wks old), tumor lesions from three hemizygous MYCN-Tg mice (9 or 10 wks old) and three homozygous MYCN-Tg mice (6 or 6.1 wks old), and ganglia from three wild-type mice (1.6 or 2 wks old). (b) SGO1 mRNA (left upper : semi-quantitative, left bottom : quantitative qPCR analysis) and protein (right) levels in neuroblastoma cell lines.

Figure 2. SGO1 is a potential transcriptional target of MYCN.

(a) MYCN overexpression induced SGO1 mRNA (left) and protein (right) expression in SH-EP cells (MYCN single-copy neuroblastoma cell line). (b) SGO1 protein levels were reduced in MYCN-downregulated IMR32 cells, but the SMC3 protein level was unchanged in these cells. Samples were harvested 3 days after non-target or MYCN-targeted shRNA lentivirus infection. Quantification of proteins on Western blots using ImageJ software. (c) Positions of E-box sequences associated with SGO1. Black circles, E-boxes; white boxes, 5′- or 3′-UTRs; gray boxes, exons. (d) ChIP analysis revealed enrichment of MYCN in E-box1 and E-box2 upstream of SGO1. A sequence 20 kb upstream of the SGO1 gene was used as a negative control. Data show the percentage of target DNA precipitated with control IgG or MYCN antibody, and are expressed as the means ± SE of at least three independent experiments.

Figure 3. SGO1 knockdown and MYCN overexpression induce G2/M accumulation.

(a) SGO1 knockdown inhibited cell proliferation only in MYCN-overexpressing SH-EP cells. The number of the cells was counted 6 days after non-target or SGO1-targeted shRNA lentivirus infection. Upper panel, relative viability; lower panel, SGO1 knockdown efficiency. Data are expressed as the mean ± SE of at least three independent experiments. (b) SGO1 knockdown inhibited cell proliferation only in MYCN-amplified neuroblastoma cell lines. Number of cells counted at 4, 5, 6, 7, or 8 days after non-target or SGO1-targeted shRNA lentivirus infection. Upper panel, relative viability; bottom panel, SGO1 knockdown efficiency. (c) Flow cytometry profiles of control and SGO1-knockdown cells at 48 hrs after non-target or SGO1-targeted shRNA lentivirus infection. Lower panel shows SGO1 knockdown efficiency.

Figure 4. SGO1 knockdown and MYCN overexpression induce DNA damage that is repaired primarily by NHEJ.

(a) Immunofluorescence of γ-H2AX and DAPI staining of IMR32 (MYCN-amplified) and SK-N-AS (MYCN-single copy) cells infected with non-targeting or SGO1-specific shRNA. Images were acquired 3 days after infection. (b) Percentage of γ-H2AX-positive cells in the cell lines shown in (a), as well as in NB39 (MYCN-amplified) and SH-EP (MYCN-single copy) neuroblastoma cells. Data represent means ± SE of three independent repeats. (c) DSB formation in SGO1-knockdown MYCN-overexpressing U2OS cells expressing EYFP-53BP1 and ECFP-Geminin 48 hrs after virus infection. Nocodazole (0.9 μg/ml) was added for 16 hrs to arrest cells in G2/M phase, and cells were counterstained with DAPI. (d) Percentage of 53BP1-positive cells relative to the total number of Geminin-positive cells in (c). Data are expressed as the mean ± SE of four independent experiments. (e) NHEJ efficiency in SGO1-knockdown MYCN-overexpressing H1299dA3-1 #1 cells and HR efficiency in SGO1-knockdown MYCN-overexpressing DR-U2OS cells. NU7026 (a DNA-PK inhibitor) was used as a negative control for the NHEJ assay. shBRCA1 and shBRCA2 were used as a negative control for the HR assay.

Figure 5. SGO1 knockdown together with MYCN overexpression induces senescence-like phenotype, but not apoptosis.

(a) TUNEL staining of control or SGO1-knockdown IMR32 cells 6 days after lentivirus infection. (b) MYCN-overexpressing or control SH-EP cells infected with lentiviral vectors expressing shRNA against SGO1 were assayed for SA-β-gal activity 6 days after infection. (c) The percentage of SA-β-gal-positive cells shown in (b). Values represent the mean ± SE of three fields. (d) Relative expression of p16 and p21 in the cells shown in (b). Values are expressed as the mean ± SE.

Figure 6. Schematic model of the synergistic phenotype of SGO1 knockdown and MYCN overexpression.