Significance of cyclin D1 overexpression in progression and radio-resistance of pediatric ependymomas

Abstract

Due to the limited efficacy of chemotherapy, the applications of adjuvant irradiation play an important role for ependymoma treatment. However, in the young ages, the resistance of residual and recurrent tumor, and long-term intellectual sequelae remain the major obstacles of radiotherapy. Understanding the mechanism of therapeutic failure caused by radio-resistance is, therefore, crucial in ependymoma treatment. Here we retrospectively analyze clinic-pathological factors in 82 cases of ependymoma less than 20 years old and identify radio-resistant genes through gene expression microarray followed by qRT-PCR validation and immunohistochemistry staining. Thirty-one out of 82 (37.8%) patients are under 3-year-old. The 10 years PFS and OS are 38% and 60%. Gross-total resection is the single significant prognostic factor for longer 10 years PFS and OS in the multivariant analysis (p<0.05). According to the microarray analysis, CCND1 is up-regulated in supratentorial and infratentorial ependymomas and is associated with DNA repair. We demonstrated that 24 primary and 16 recurrent ependymomas were up-regulated, and 5 out of 7 paired samples exhibited higher CCND1 expression in recurrent tumors. We also found RAD51, another DNA repair gene, was up-regulated in supratentorial and infratentorial ependymomas. Knocking down CCND1 reduced cell proliferation and repressed several genes associated with S-phase and DNA repair. Homologous recombination activities of DNA repair were significantly decreased in CCND1-deficient cells while the level of γH2AX was increased after irradiation. In summary, these observations suggest a robust role of CCND1 in regulating cell proliferation and radio-resistance in ependymomas, providing a potential therapeutic target for pediatric ependymomas.

Keywords: CCND1; ependymoma; pediatric; radio-resistance.

Figure 1. Clinical factors for Survival and Progression

(A-B) The 10 years overall survival (A) and progression-free-survival (B) of different locations were distinct. Red line represents infratentorium. Green line represents spine. Blue line represents supratentorium. (C) The 10 years overall survival for more than or equal to 3 vs. less than 3-year-old patients were no significant (P=0.844). Red line represents equal or more than 3-year-old. Blue line represents less than 3-year-old. (D) The age less than 3-year-old was significantly related to worse 10 years progression-free survival (P=0.040). Red line represents equal or more than 3-year-old. Blue line represents less than 3-year-old. (E) The gross total resection was significantly related to a longer 10 years progression-free survival (P=0.003). Red line represents gross-total resection. Blue line represents non-total resection. (F) There is no significant difference of 10 years progression-free survival among adjuvant treatment by using Kaplan Meier method (p=0.63). Red line represents adjuvant radiation alone (N=41). Green line represents adjuvant chemo-radiation (N=39). Blue line represents none or chemotherapy alone (N=20).

Figure 2. Differentiated genes in infratentorium and supratentorium ependymomas

(A-B) Venn diagram show the up-regulated (A) (q <0.05, Fold change ≧ 2) or down-regulated (B) (q <0.05, Fold change ≦ 2) genes in supratentorium (left) and infratentorium (right) compared with normal tissues. The numbers of the intersections indicate the overlapped genes across all groups. (C) Up-regulated (left panel) and down-regulated (right panel) genes in supratentorium and infratentorium were subjected to Gene Ontology (GO) database searches. (D) Schematic representation for identifying DNA damage associated targets. The putative targets were obtained from up-regulated or down-regulated genes which are associated with DNA damage response. (E) Heatmap showed that up-regulated genes were associated with DNA damage response and sorted by fold change. (left: infratentorium, right: supratentorium).

Figure 3. CCND1 and RAD51 are overexpressed in primary and recurrent ependymomas

(A-B) CCND1 expression validated in primary (Pre-RT) and recurrent (Post-RT) supratentorium (A) and infratentorium (B) compared with normal whole brain tissue and cerebellum, respectively. qRT-PCR results are presented as mean±SD for duplicate samples. (C) CCND1 expression was more in primary and recurrent supratentorium than infratentorium. ^*p<0.05 by t-test. (D-E) RAD51 expression validated in primary and recurrent supratentorium (D) and infratentorium (E) compared with normal whole brain tissue and cerebellum, respectively. qRT-PCR results are presented as mean±SD for duplicate samples. (F) RAD51 expression was more in primary supratentorium than infratentorium. ^*p<0.05 by t-test. (G-H) IHC analyses confirmed protein levels of CCND1 (G) and RAD51 (H) in supratentorium and infratentorium. ST: supratentorium, PF: infratentorium. Scale bar: 100 μm.

Figure 4. CCND1 and RAD51 are overexpressed in paired recurrent ependymomas

(A-B) qRT-PCR analyses confirmed CCND1 (ST: upper panel, PF: left panel) and RAD51 (ST: lower panel, PF: right panel) expression in paired samples (pre-RT v.s. post-RT) from supratentorium (A) and infratentorium (B). ST: supratentorium, PF: infratentorium. (C) IHC analyses confirmed protein levels of CCND1 in paired samples (pre-RT vs. post-RT). Scale bar: 100 μm.

Figure 5. CCND1 regulate cell proliferation and DNA repair in ependymomas

(A) Knocking downCCND1 (shCCND1) expression in ependymoma cells, which was confirmed through qRT-PCR and immunoblotting. (B) Knocking down CCND1 expression decreasedcell proliferation rate in ependymoma cells asmeasured by MTT assay. (C) Significant suppression of downstream genes was validated by qRT-PCR. qRT-PCR results are presented as mean±SD for duplicate samples.^**p<0.01 by t-test. (D) Cell proliferation rate were measured after radiation treatment (6Gy). (E) Strong γH2AX expression level indicates DNA damage after radiation treatment, and be enhanced in shCCND1. (F) Knocking down CCND1 decreased DNA repair in ependymoma cells after radiation treatment (6 Gy and 8Gy) as measured by homologous recombination assay. (G) CCND1 expression were measured after radiation treatment (6 Gy and 8 Gy). qRT-PCR results are presented as mean±SD for duplicate samples.^**p<0.05 by t-test.

Figure 6. Palbociclib treatment regulate cell proliferation in ependymomas

(A) Palbociclib treatment decreasedcell proliferation rate in ependymoma cells asmeasured by MTT assay. (B) Flow cytometry showed accumulated cells in the G1 phase after palbociclib treatment. (C) Palbociclib treatment decreased RB phosphorylation in ependymomas. (D) Significant suppression of downstream genes was validated by qRT-PCR. qRT-PCR results are presented as mean±SD for duplicate samples.^**p<0.01 by t-test. (E) Prolonged γH2AX expression level indicates DNA damage after radiation treatment, and be enhanced in palbociclib treatment.