Nuclear NAD+ synthase nicotinamide mononucleotide adenylyltransferase 1 contributes to nuclear atypia and promotes glioma growth

Abstract

Background: Glioma is a malignant primary brain tumor with a poor prognosis and short survival. NAD⁺ is critical for cancer growth; however, clinical trials targeting NAD⁺ biosynthesis had limited success, indicating the need for mechanistic characterization. Nuclear atypia, aberrations in the size and shape of the nucleus, is widely observed in cancer and is often considered a distinctive feature in diagnosis; however, the molecular underpinnings are unclear.

Methods: We carried out high-resolution immunohistochemical analyses on glioma tissue samples from 19 patients to analyze the expression of NAD⁺ synthase nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1), and its correlation with nuclear atypia in gliomas. Utilizing a Drosophila model of glial neoplasia, we investigated the genetic role of nuclear NMNAT in glioma growth in vivo, elucidating the cellular mechanisms of NMNAT1 in promoting nuclear atypia and glioma growth.

Results: In low-grade glioma and glioblastoma, a higher transcription level of NMNAT1 is correlated with poorer disease-free survival. Samples of high-grade gliomas contained a higher percentage of glial cells enriched with NMNAT1 protein. We identified a specific correlation between nuclear NMNAT1 protein level with nuclear atypia. Mechanistic studies in human glioma cell lines and in vivo Drosophila model suggest that NMNAT1 disrupts the integrity of the nuclear lamina by altering the distribution of lamin A/C and promotes glioma growth.

Conclusions: Our study uncovers a novel functional connection between the NAD⁺ metabolic pathway and glioma growth, reveals the contribution of the NAD⁺ biosynthetic enzyme NMNAT1 to nuclear atypia, and underscores the role of nuclear NMNAT1 in exacerbating glioma pathology.

Keywords: Drosophila; EGFR; NAD+; glioblastoma; lamin A/C.

Figure 1.

Correlation of nicotinamide mononucleotide adenylyltransferase (NMNAT) expression levels with patient survival in glioma. (A and B) Visualization of NMNAT1 and NMNAT2 mRNA expression in glioma from GEPIA datasets. The expression data are first log₂ (TPM + 1) scaled and the log₂FC is defined as median (tumor) – median (normal). Overexpressed genes as those passing the following thresholds: log₂FC > 1, percentage > 0.9. Dot plot of expression of NMNAT in normal samples (n = 207), LGG (low-grade glioma) (n = 518), and GBM (glioblastoma) (n = 163). (C and D) Survival curves for patients with LGG expressing NMNA1 (C) and NMNAT2 (D) (top 50% versus bottom 50%). (E and F) Survival curves for patients with GBM expressing NMNA1 (E) and NMNAT2 (F) (top 50% versus bottom 50%). (G and H) Survival curves for patients with GBM are shown, with tumors expressing NMNA1 (G) and NMNAT2 (H) (top 10% versus bottom 10%). All associated log-rank P-values are shown. High NMNAT1-expressing tumors have extremely poor relapse-free rates compared to low NMNAT1-expressing tumors. In contrast, high NMNAT2 expression has been shown to better prognose in LGG but not GBM.

Figure 2.

The expression of nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) protein is increased in GBM. (A) Collection of human glioma pathological samples with different grades and glioma types. (B and C) Immunohistochemistry detection of NMNAT1 in low-grade glioma (B) and GBM (C). The high magnification of the NMNAT1 staining nucleus shows the box area with the white dashed line on the left side. The white arrows indicate the negative staining nucleus and the yellow arrows indicate the nucleus with positive staining. Scale bar, 50 µm. (D) Quantification of the percentage of NMNAT1 positive staining in the nucleus in A. Data are presented as mean ± SD n = 19. The significance level was established by t-test. *P ≤ .05.

Figure 3.

The expression of nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) increased in glioma compared to adjacent normal tissue. (A) Overview of H&E detection for human glioma sample with adjacent normal tissue. The normal tissue is marked with black dashed lines and tumor tissue is marked with red dashed lines, respectively. Scale bars, 1000 µm. (B) A high magnification area of H&E staining from the black box area in the first column. The second column shows the high magnification of the white box. Scale bars, 100 µm. (C) Quantification of the cell density of normal tissue and tumor tissue. Data are presented as paired, n = 3. (D) Overview of Immunohistochemistry detection of NMNAT1 in human glioma sample with adjacent normal tissue. The normal tissue is marked with black dashed lines and tumor tissue is marked with red dashed lines, respectively. Scale bars, 1000 µm. (E) A high magnification area of NMNAT1 staining from the black box area in the first column. The second column shows the high magnification of the white box. Scale bars, 100 µm. (F) Quantification of the percentage of NMNAT1 positive staining in adjacent tissue and GBM. n = 3. The significance level was established by t-test. **P ≤ .01.

Figure 4.

Reduced nuclear circularity and increased nucleus size are observed in GBM, and NMNAT1 expression is correlated with nuclear circularity in human gliomas. (A, C, and D) Quantification of nuclear circularity for gliomas in individual samples. Nucleus numbers are listed. Samples are aligned by median value and grouped into GBM and astrocytoma (A), oligodendroglioma (C), and ependymoma (D). (B) Quantification of nuclear circularity for astrocytoma and GBM. The significance level was established by the Wilcoxon rank sum test. (E, G, and H) Quantification of the nucleus size in GBM and astrocytoma (E), oligodendroglioma (G), and ependymoma (H). (F) Quantification of nucleus size for astrocytoma and GBM. The significance level was established by the Wilcoxon rank sum test. (I–K) Quantification of nuclear circularity and NMNAT1 intensity for each nucleus in individual samples. Samples are aligned by the median value of circularity and grouped into GBM and astrocytoma (I), oligodendroglioma (J), and ependymoma (K). (L) The linear fit of NMNAT1 intensity and nuclear circularity of gliomas. (M) The linear regression of NMNAT1 intensity and nuclear circularity of GBM and astrocytoma. (N and O) The linear regression of NMNAT1 intensity and nuclear circularity of oligodendroglioma (N) and ependymoma (O). ***P ≤ .001. ****P ≤ .0001.

Figure 5.

Overexpression of nuclear nicotinamide mononucleotide adenylyltransferase (NMNAT) promotes glial neoplasia growth in Drosophila. (A) Brains at 90% pupal stage with the glial expression of lacZ + Egfr, nucNMNAT + Egfr, cytNMNAT + Egfr, and nucNMNAT^WR + Egfr were probed for Repo (green), F-actin (red), and DAPI (blue). The second row shows the high magnification of white dashed box areas in the first row. The Glial neoplasia area is marked with cyan dashed lines. Scale bars, 30 µm. (B) Quantification of volumes of glial neoplasia with glial expression of lacZ + Egfr, nucNMNAT + Egfr, cytNMNAT + Egfr, and nucNMNAT^WR + Egfr. Data are presented as mean ± SD, n ≥ 7. The significance level was established by one-way ANOVA post hoc Bonferroni test. (C) The eclosion rate of flies with glial neoplasia. (D) Lifespan for each group. The significance level was established by a log-rank test. (E) Quantification of average climbing speed for each fly. Data are presented as mean ± SD, n = 7. The significance level was established by one-way ANOVA post hoc Bonferroni test. *P ≤ .05. **P ≤ .01. ***P ≤ .001. ****P ≤ .0001.

Figure 6.

Overexpression of nuclear nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) leads to nuclear atypia by altering lamin A/C localization. (A) Brains at 90% pupal with the glial expression of lacZ + Egfr, nucNMNAT + Egfr, cytNMNAT + Egfr, and nucNMNAT^WR + Egfr were probed for Repo (green), F-actin (red), and DAPI (blue). The second row shows the high magnification of white box areas in the first row. Glial neoplasia is marked with dashed lines. The third row shows the high magnification of the glial neoplasia area in the second row. Scale bars, 30 µm. (B and C) Quantification of nucleus size (B) and nuclear circularity (C) for each cell in the glial neoplasia area with the glial expression of lacZ + Egfr, nucNMNAT + Egfr, cytNMNAT + Egfr, and nucNMNAT^WR + Egfr. Data are presented as median ± quarter, n ≥ 3. The significance level was established by one-way ANOVA post hoc Bonferroni test. (D) Human GBM T98G cells transfected with DsRed-NMNAT1 were stained for DAPI (blue) and Lamin A/C (green). (E–H) The high magnification of the yellow boxed area (E and G) and white boxed area (F and H) in D. The intensity (0-4095) of lamin A/C is indicated in a heat map (I). (D–M) Scale bars, 10 µm. (J and K) The high magnification of the yellow (J) and white boxed (K) area in I. (L and M) The surface plot of J and K. Quantification of nucleus size (N), nuclear circularity (O), lamin A/C intensity (P). Data are presented as mean ± SD, n ≥ 20. The significance level was established by t-test. *P ≤ .05. **P ≤ .01. ****P ≤ .0001.