Brachyury gene copy number gain and activation of the PI3K/Akt pathway: association with upregulation of oncogenic Brachyury expression in skull base chordoma

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OBJECTIVE

Chordoma is a slow-growing but clinically malignant tumor, and the prognosis remains poor in many cases. There is a strong impetus to develop more effective targeted molecular therapies. On this basis, the authors investigated the potential of Brachyury, a transcription factor involved in notochord development, as a candidate molecular target for the treatment of chordoma.

METHODS

Brachyury gene copy number and expression levels were evaluated by quantitative polymerase chain reaction in 27 chordoma samples, and the transcriptomes of Brachyury high-expression tumors (n = 4) and Brachyury low-expression tumors (n = 4) were analyzed. A chordoma cell line (U-CH2) was used to investigate the signaling pathways that regulate Brachyury expression.

RESULTS

All chordoma specimens expressed Brachyury, and expression levels varied widely. Patients with higher Brachyury expression had significantly shorter progression-free survival (5 months, n = 11) than those with lower expression (13 months, n = 16) (p = 0.03). Somatic copy number gain was confirmed in 12 of 27 (44%) cases, and copy number was positively correlated with Brachyury expression (R = 0.61, p < 0.001). Expression of PI3K/Akt pathway genes was upregulated in Brachyury high-expression tumors, and suppression of PI3K signaling led to reduced Brachyury expression and inhibition of cell growth in the U-CH2 chordoma cell line.

CONCLUSIONS

Activation of the PI3K/Akt pathway and Brachyury copy number gain are strongly associated with Brachyury overexpression, which appears to be a key event in chordoma growth regulation. These findings suggest that targeting Brachyury and PI3K/Akt signaling may be an effective new approach for treating chordoma.

ABBREVIATIONS cDNA = complementary DNA; DMSO = dimethyl sulfoxide; EMT = epithelial-mesenchymal transition; FC = fold change; hESC = human embryonic stem cell; KEGG = Kyoto Encyclopedia of Genes and Genomes; mTOR = mammalian target of rapamycin; PFS = progression-free survival; qRT-PCR = quantitative real-time reverse transcription polymerase chain reaction.

Article Information

Correspondence Akitake Mukasa, Department of Neurosurgery, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. email: mukasa-nsu@umin.ac.jp.

INCLUDE WHEN CITING Published online July 28, 2017; DOI: 10.3171/2016.12.JNS161444.

Disclosures The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Chart showing characteristics of 19 patients with skull base chordoma. Age denotes the patient’s age when the surgery was performed, and PFS consists of the period from the date of surgery to the date when the recurrence was detected. The asterisks indicate that the RNA or DNA was of suitable quality to be analyzed. For the time course, the black circles indicate surgery (analyzed sample); white circles denote other surgeries; triangles represent radiation therapy or Gamma Knife surgery; “x” denotes patient death; and “+” denotes time of latest follow-up. GTR = gross-total resection; PR = partial resection.

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    A: Distribution of Brachyury expression levels in all chordoma specimens (n = 27) measured by qRT-PCR. Box plot shows maximum, upper quartile, median, lower quartile, and minimum. Expression levels are relative to that of normal brain. B: The relationship between Brachyury gene expression and Ki-67 labeling index. The coefficient of correlation was 0.47. C: Kaplan-Meier curves of PFS comparing the Brachyury high-expression and Brachyury low-expression groups. The Brachyury high-expression group had significantly shorter PFS than the Brachyury low-expression group (p = 0.03).

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    A: TaqMan Copy Number Assays targeting exon 7 of the Brachyury gene revealed that the Brachyury gene copy number in chordoma was significantly higher than in matched normal blood samples (n = 27, p < 0.001). B: There was a significant positive correlation between Brachyury gene copy number (exon 7) and Brachyury expression (R = 0.61, p < 0.001). C: Representative positive and negative immunostaining of phosphorylated S6 ribosomal protein is shown. The samples containing cells with stained cytoplasm were considered positive.

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    Effect of PI3K pathway inhibition on Brachyury expression and chordoma cell growth. A: Western blot analysis of Brachyury, phospho-Akt (p-Akt), Akt, or β-actin in U-CH2 cells treated with 0.1 μM BEZ235, 1 μM BEZ235, or DMSO. The inhibition of the PI3K/Akt pathway by BEZ235 was confirmed by suppression of phospho-Akt expression. Phospho-Akt and Brachyury expression were decreased after inhibition of the PI3K/Akt pathway by BEZ235. B: The chordoma cell line U-CH2 was treated with PI3K inhibitors (LY294002, BEZ235, or wortmannin), mTOR inhibitor (rapamycin), or vehicle (DMSO) for 24 hours. Brachyury expression was measured by qRT-PCR, and expression levels were compared with control (*p < 0.05). C: The U-CH2 cell line was treated with lipofectamine 2000 (control), 5 pmol of small interfering RNA targeting Brachyury (Bra1 or Bra3), or Negative Control Medium GC (Nega M). Suppression of Brachyury expression was confirmed by qRT-PCR. D: The U-CH2 cell growth curves were evaluated by WST-1 assay. Cell growth decreased in response to Brachyury suppression. E: Cell growth curves of U-CH2 cells treated with 0.1 μM BEZ235, 1 μM BEZ235, or DMSO evaluated by WST-1 assay. Maximum significant cell growth inhibition was achieved with 0.1 μM BEZ235. Error bars in each figure show the standard deviation.

  • View in gallery

    Schematic depiction of the interplay between Brachyury copy number gain, Brachyury overexpression, and activation of the PI3K/Akt pathway in chordoma pathogenesis.

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