Coexpression of neuronatin splice forms promotes medulloblastoma growth

Abstract

Medulloblastoma (MB) is the most common pediatric brain cancer. Several important developmental pathways have been implicated in MB formation, but fewer therapeutic targets have been identified. To locate frequently overexpressed genes, we performed a comprehensive gene expression survey of MB. Our comparison of 20 primary tumors to normal cerebellum identified neuronatin (NNAT) as the most frequently overexpressed gene in our analysis. NNAT is a neural-specific developmental gene with alpha and beta splice forms. Functional evaluation revealed that RNA interference knockdown of NNAT causes a significant decrease in proliferation. Conversely, coexpression of both splice forms in NNAT-negative MB cell lines increased proliferation, caused a significant shift from G(1) to G(2)/M, and increased soft agar colony formation and size. When expressed individually, each NNAT splice form had much less effect on these in vitro oncogenic predictors. In an in vivo model, the coexpression of both splice forms conferred the ability of xenograft formation to human MB cells that do not normally form xenografts, whereas a control gene had no effect. Our findings suggest that the frequently observed overexpression of both NNAT splice forms in MB enhances growth in this cancer.

Fig. 1

Neuronatin (NNAT) is overexpressed in medulloblastoma (MB) tumors. (A) Reverse transcriptase–PCR was performed on a panel of MB resections (1–20; top lanes), xenografts, cell lines, and normal tissues (bottom lanes: lanes 1–3, cerebellum; 4, gray matter; 5, white matter; 6, caudate nucleus; 7, fetal brain; 8, total normal brain; 9 and 10, cerebellum; 11, fetal brain). NNATα is present at levels equal to or higher than those of NNATβ in MBs, xenografts, and cell lines. In fetal brain and in cerebellum, NNATβ is present at levels equal to or higher than those of NNATα. In the other normal brain structures we tested, both NNAT splice forms are absent. (B) Immunohistochemical staining of a primary MB with anti-NNAT (left) or without anti-NNAT (right). NNAT is overexpressed in MBs at the protein level and appears to localize in the membrane or cytoplasm in most cells, as shown in the inset, although some cells show a nuclear localization. Original magnification, ×100. (C) Semiquantitative PCR of MBs, which express a range of Serial Analysis of Gene Expression (SAGE) transcript levels (indicated above the corresponding set of PCRs for each tumor) performed at 24, 27, 30, and 33 cycles (indicated by 1, 2, 3, and 4, respectively, below gel). Higher overall NNAT expression correlates with reexpression of NNATα.

Fig. 2

RNA interference (RNAi)-mediated knockdown of neuronatin (NNAT) splice forms. (A) Western blot of D283Med cells 48 h postelectroporation of dicer-ready RNA interference duplexes targeting NNATα only or NNATα+β; equal amounts of protein were loaded per well. neg, scrambled RNAi negative control. (B) alamarBlue proliferation assay of D283Med cells after small interfering RNA electroporation. Knockdown of the NNATα splice form alone or NNATα+β splice forms results in a significant decrease in proliferation. Error bars represent variation in triplicate wells. RFU, relative fluorescence units.

Fig. 3

Analysis of the growth properties of cells expressing neuronatin (NNAT) splice forms. (A) Expression of NNATα+β causes a shift from G₁ to G₂M, whereas expression of NNATα or NNATβ alone did not significantly alter cell cycle progression. (B) Growth curves of UW228 medulloblastoma (MB) cell lines expressing pLAPSN, NNATα, NNATβ, and NNATα+β. Cells expressing NNATα and NNATβ grew at roughly similar rates, whereas the cell line expressing NNATα+β grew at a significantly increased rate; cells expressing the control pLAPSN grew the slowest. A representative experiment is shown, with error bars representing the variation in triplicate wells. (C) Coexpression of NNATα and NNATβ causes significant anchorage-independent growth in soft agar. UW228 MB cell lines expressing the control pLAPSN or NNATβ formed few colonies, which were mostly 0–0.25 μm in diameter. The expression of NNATα slightly increased both the number and size of colonies formed, but the coexpression of NNATα and NNATβ together caused a significant increase in both the number and size of soft agar colonies.

Fig. 4

Expression of neuronatin (NNAT) α and β splice forms in UW228 medulloblastoma cells induces subcutaneous tumor formation in the flanks of three of three athymic nude mice. (A) Hematoxylin- and eosin-stained sections of a xenograft expressing NNATα+β showing a moderately cellular tumor with pleomorphic nuclei. (B) Hematoxylin- and eosin-stained sections of a xenograft expressing NNATα+β with increased proliferation of malignant cells as indicated by the presence of mitotic figures (arrows).