Tuberin Negatively Promotes ASAH1/SPHK1/S1PR3 Axis Mediated Tumorigenesis in Lymphangioleiomyomatosis (LAM)

Abstract

Rationale. Lymphangioleiomyomatosis (LAM) is a progressive pulmonary disease that exclusively affects women,caused by defects, or mutations, on two genes-tuberous sclerosis complex 1 and 2 (TSC1 and TSC2), and product the proteins hamartin and tuberin, respectively. LAM is typically caused by TSC2 mutations resulting in mTORC1 activation. Treatment with Rapamycin in clinical trial suppressed tumors growth somewhat but tended to increase in volume after drug withdrawal. Sphingolipids have been shown to play a key role in the regulation of cell growth and proliferation in cancer. We have recently shown that sphingolipids metabolism are involved in LAM in vitro and in vivo. Methods. Sphingolipids metabolism related enzyme was identified by metabolomics profiling, and expressions of acid ceramidase (ASAH1) and sphingosine kinase 1 (SPHK1) were characterized, in LAM patients tissues and TSC2-deficient cells, by western blotting and real time RT-PCR analysis. Cell migration was determined in Boyden chambers, by using the selective ASAH1 or SPHK1 antagonist and silencing of them expressions with shRNA. Sphingosine-1-phosphate level in the plasma from LAM and normal patients or cell culture were detected by human S1P ELISA kit. S1PR3 was screened by real time RT-PCR and cell viability and death detected by MTT assay and PI/CV using the selective S1PR3 antagonist and small interfering RNA. Results. Expressions of ASAH1 and SPHK1 were significantly higher in LAM lung tissues than normal patients and TSC2-deficient cells relative to TSC2-addback cells. TSC2 negatively regulated the expression of ASAH1 and SPHK1 in a rapamycin-insensitive manner, and the ASAH1 inhibitor ARN14976 (17a) and the SPHK1 antagonist PF543, potently suppressed the viability of TSC2-deficient cells. ASAH1 and SPHK1 knockdown led to reduced viability, migration and invasion in TSC2-deficient cells. Meanwhile, we investigated that the effects of S1P were involvement of S1PR3, and the viability and protein expression of the TSC2-null cells were markedly reversed by treatment of TY52156, a selective antagonist for S1PR3, and S1PR3-siRNA. Conclusions. These results identify a novel finding of rapamycin-insensitive signature of disorder of sphingolipid metabolism in TSC2-null cells and validate novel hypothesis that TSC2 regulates sphingolipid productions and actions via ASAH1/SPHK1/S1PR3 axis in tumorigenesis. Targeting abnormal sphingolipid metabolism may have therapeutic value in LAM and TSC-related diseases, and possibly in other conditions associated with mTOR hyperactivation.