Rapamycin-insensitive up-regulation of adipocyte phospholipase A2 in tuberous sclerosis and lymphangioleiomyomatosis.

Chenggang Li,Yanan Guo,Kai-Feng Xu,Ivan O Rosas,David J Kwiatkowski,Jane J Yu,Yang Sun,Erik Zhang,Juan C Osorio,Yongzhong Zhan,Po-Shun Lee,David J Reiner

doi:10.1371/journal.pone.0104809

Abstract

Tuberous sclerosis syndrome (TSC) is an autosomal dominant tumor suppressor gene syndrome affecting multiple organs, including renal angiomyolipomas and pulmonary lymphangioleiomyomatosis (LAM). LAM is a female-predominant interstitial lung disease characterized by the progressive cyst formation and respiratory failure, which is also seen in sporadic patients without TSC. Mutations in TSC1 or TSC2 cause TSC, result in hyperactivation of mammalian target of rapamycin (mTOR), and are also seen in LAM cells in sporadic LAM. We recently reported that prostaglandin biosynthesis and cyclooxygenase-2 were deregulated in TSC and LAM. Phospholipase A2 (PLA2) is the rate-limiting enzyme that catalyzes the conversion of plasma membrane phospholipids into prostaglandins. In this study, we identified upregulation of adipocyte AdPLA2 (PLA2G16) in LAM nodule cells using publicly available expression data. We showed that the levels of AdPLA2 transcript and protein were higher in LAM lungs compared with control lungs. We then showed that TSC2 negatively regulates the expression of AdPLA2, and loss of TSC2 is associated with elevated production of prostaglandin E2 (PGE2) and prostacyclin (PGI2) in cell culture models. Mouse model studies also showed increased expression of AdPLA2 in xenograft tumors, estrogen-induced lung metastatic lesions of Tsc2 null leiomyoma-derived cells, and spontaneous renal cystadenomas from Tsc2+/− mice. Importantly, rapamycin treatment did not affect the expression of AdPLA2 and the production of PGE2 by TSC2-deficient mouse embryonic fibroblast (Tsc2 −/−MEFs), rat uterine leiomyoma-derived ELT3 cells, and LAM patient-associated renal angiomyolipoma-derived “mesenchymal” cells. Furthermore, methyl arachidonyl fluorophosphate (MAFP), a potent irreversible PLA2 inhibitor, selectively suppressed the growth and induced apoptosis of TSC2-deficient LAM patient-derived cells relative to TSC2-addback cells. Our findings suggest that AdPLA2 plays an important role in promoting tumorigenesis and disease progression by modulating the production of prostaglandins and may serve as a potential therapeutic target in TSC and LAM.

Highlights

Tuberous sclerosis syndrome (TSC) is an autosomal dominant tumor suppressor gene syndrome characterized by neurologic disease, benign tumors in multiple organs, including renal angiomyolipomas, and pulmonary lymphangioleiomyomatosis (LAM), which is due to inactivating mutations in either TSC1 or TSC2 [1]
Since prostaglandin biosynthesis is initiated by phospholipase A2 (PLA2) acting on membrane phospholipids, we examined its expression using public available expression array data sets from pulmonary LAM nodule cells collected by laser-capture microdissection (GEO data set GSE12027) [27], lung cancer/tumor (GEO data set GSE10072 and GSE19804) [25,34], and control lungs (GEO data set GSE10072 and GSE19804) [25,34] (Figure 1A)
Confocal microscopy showed that smooth muscle actin-positive cells were stained with AdPLA2, whereas pulmonary artery cells were negative with AdPLA2 staining (Figure 2C)

Summary

Introduction

Tuberous sclerosis syndrome (TSC) is an autosomal dominant tumor suppressor gene syndrome characterized by neurologic disease, benign tumors in multiple organs, including renal angiomyolipomas, and pulmonary lymphangioleiomyomatosis (LAM), which is due to inactivating mutations in either TSC1 or TSC2 [1]. Loss of either TSC1 or TSC2 leads to increased Rheb activity which promotes mTORC1 activity and downstream phosphorylation of S6K, S6 and 4E-BP1, leading to increased protein synthesis for cellular growth and metabolism [4] This understanding led to multiple preclinical studies which demonstrated the effectiveness of rapamycin, an mTORC1 inhibitor, in multiple animal models of TSC [5,6,7,8,9,10,11], which led to rapid clinical translation, and demonstration that rapamycin has clinical benefit for TSC kidney, lung, and brain tumors [12,13,14,15]. We reported that TSC2 negatively regulates COX-2 expression, prostaglandin production and tumorigenesis in an mTORC1insensitive, but mTORC2-dependent manner [10]

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