Reduced activity of CD13/aminopeptidase N (APN) in aggressive meningiomas is associated with increased levels of SPARC

Abstract

Meningiomas are the second most common brain tumors in adults, and meningiomas exhibit a tendency to invade adjacent structures. Compared with high-grade gliomas, little is known about the molecular changes that potentially underlie the invasive behavior of meningiomas. In this study, we examined the expression and function of the membrane alanyl-aminopeptidase [mAAP, aminopeptidase N (APN), CD13, EC3.4.11.2] zinc-dependent ectopeptidase in meningiomas and meningioma cell lines, based on its prior association with tumor invasion in colorectal and renal carcinomas. We found a significant reduction of APNmRNA and protein expression, as well as enzymatic activity, in high-grade meningiomas. While meningioma tumor cell proliferation was not affected by either pharmacologic APN inhibition or siRNA-mediated APN silencing, APN pharmacologic and siRNA knockdown significantly reduced meningioma cell invasion in vitro. Next, we employed pathway-specific cDNA microarray analyses to identify extracellular matrix and adhesion molecules regulated by APN, and found that APN-siRNA knockdown substantially increased the expression of secreted protein, acidic and rich in cysteine (SPARC)/osteonectin. Finally, we demonstrated that SPARC, which has been previously associated with meningioma invasiveness, was increased in aggressive meningiomas. Collectively, these results suggest that APN expression and enzymatic function is reduced in aggressive meningiomas, and that alterations in the balance between APN and SPARC might favor meningioma invasion.

Figure 1

Expression of CD13/aminopeptidase N (APN) in human meningiomas. A. Immunodetection of APN in paraffin‐embedded tumor samples of meningiomas with different grades of malignancy. Strong predominantly membrane‐bound immunoreaction for APN is seen in meningothelial (upper‐left figure) and fibroblastic World Health Organization (WHO) grade I meningiomas. In aggressive WHO grade II (upper‐right figure) or WHO grade III (lower panel) meningiomas, the membrane‐bound APN staining is restricted to a few cells (lower‐left figure, arrows) or cell groups (lower‐right figure). Bars represent 100 µm. Inset shows liver tissue as a positive control. B. Expression of APN mRNA as measured by real‐time polymerase chain reaction (PCR) is decreased in both atypical (grade II) and anaplastic (grade III) meningiomas (*P < 0.05). Values are normalized to α‐tubulin expression; mean ± standard error of the mean (SEM) are shown. C. Enzymatic activity is decreased especially in anaplastic grade III meningiomas (mean ± SEM; *P < 0.05). D. Western blot detection of APN from human meningioma samples confirm reduced APN protein amounts in aggressive grade II or grade III meningiomas. Densitometric values of APN normalized to GAPDH are given below. Abbreviation: OD = optical density.

Figure 2

Expression and activity of aminopeptidase N (APN) in meningioma cells. High APN expression can be found on the mRNA level (A), by Western blot analyses (B) and by measuring the enzymatic activity (C), especially in benign meningioma cell lines HBL52 and Ben‐Men‐1, as well as in the malignant meningioma cell line IOMM‐Lee. D. Cells with high APN enzymatic activity (IOMM‐Lee) have higher migration rates than cells with low APN activity (KT21MG1) in a transwell migration assay. Mean ± standard deviation (SD) are shown; densitometric values are given in B. Abbreviations: GAPDH = glyceraldehyde‐3‐phosphate dehydrogenase; OD = optical density.

Figure 3

Aminopeptidase N (APN) inhibition in meningioma cells do not affect meningioma cell proliferation. A. Significant inhibition of APN mRNA (left figure; ***P < 0.01) and protein levels (right figure) by two different siRNA species. B. Both Ben‐Men‐1 and IOMM‐Lee cells have reduced APN enzymatic activity following siRNA‐mediated knockdown [mean ± standard error of the mean (SEM); *P < 0.05]. C. No substantial effects on meningioma cell proliferation are seen after inhibition by APN inhibitors (actinonin, PAQ22 or WM15) or by siRNA (mean ± SEM are shown for IOMM‐Lee cells).

Figure 4

Invasiveness of IOMM‐Lee meningioma cells is significantly inhibited by aminopeptidase N (APN)‐siRNA knockdown (A) or APN inhibition by actinonin (B) [mean ± standard error of the mean (SEM);*P < 0.05].

Figure 5

Aminopeptidase N (APN) expression in meningiomas is inversely related to SPARC expression. A. siRNA knockdown in IOMM‐Lee meningioma cells is associated with increased SPARC mRNA expression [mean ± standard error of the mean (SEM); *P < 0.05]. B. SPARC mRNA levels in six meningioma cell lines. C. APN‐siRNA‐mediated reduction of APN protein level is associated with increased SPARC protein (right figure shows normalized densitometric analyses for band intensities). D. Measuring of SPARC mRNA in human meningiomas reveals significantly increased levels in aggressive meningiomas, confirming previous immunohistochemical findings (26) (mean ± SEM; *P < 0.05). E. Association between SPARC and APN immunoexpression in grade I and grade III human meningiomas. An inverse relation is present with high SPARC but low APN expression in grade III meningiomas and vice versa in grade I meningioma. Serial sections of the same tumor region are shown. Arrows indicate retained APN staining in blood vessels in an otherwise APN‐negative meningioma. F. Scatter plot depicting the correlation between APN and SPARC mRNA expression in 16 meningiomas of different World Health Organization (WHO) grade. G. Reprobing of the APN blot (Figure 1D) with an anti‐SPARC antibody shows an inverse relation between APN and SPARC expression in the majority of tumors.