TSC2 Deficiency Increases PTEN via HIF1α

Abstract

Mutations in the tuberous sclerosis tumor suppressor genes (TSC1 and TSC2) result in autosomal dominant diseases characterized by benign tumors (hamartomas) in the kidney, heart, brain, and other organs (1). Approximately 1 in 6000 live births is linked to a germ line-inactivating mutation of either of the two genes, TSC1 or TSC2. Thus, each mutated gene accounts for ϳ50% of the cases (2). TSC2 associates with TSC1 to form the active signaling complex (3). The C terminus of TSC2 contains a GTPase-activating protein domain, which blocks mTOR activity by increasing hydrolysis of GTP associated with the small G-protein Rheb (Ras homolog enriched in the brain) (4, 5). Growth factor-stimulated Akt and other kinases phosphorylate TSC2 at specific sites, resulting in its dissociation from the TSC1/2 complex (6 -9).The lipid phosphatase activity of the tumor suppressor PTEN dephosphorylates the second messenger phosphatidylinositol (PI) 5 3,4,5-trisphosphate, thus negatively regulating the PI 3-kinase signaling pathway (10). Germ line mutations in PTEN cause cancer predisposition syndromes, such as Cowden disease (11-13). Also, loss of PTEN is common in many tumors, including sporadic glioblastoma, endometrial carcinoma, melanoma, meningioma, and renal, breast, prostate, and small cell lung cancer (11-13). Whereas PTEN deficiency predisposes to malignancy, it is rare in TSC patients (11,13,14). In PTENdeficient cancer cells, even in the absence of growth factors, Akt is constitutively active, which results in phosphorylation and inactivation of TSC2 and activation of mTOR (8, 10, 15). Similarly, the disruption of TSC2 results in significantly increased mTOR activity (16 -18). In the latter case, the mTOR activation leads to inactivation of Akt through a negative feedback loop involving IRS (insulin receptor substrate) proteins (17, 19 -21). However, additional signaling pathway(s) probably contribute to reduced Akt activation in TSC2 deficiency.In this report, we demonstrate that TSC2 deficiency results in increased expression of PTEN. As a mechanism, we show that HIF1␣ positively regulates the transcription of PTEN, using a canonical HIF-responsive element. Furthermore, we demonstrate that renal angiomyolipomas in TSC patients express elevated levels of HIF1␣ and PTEN protein. Thus, increased levels of PTEN in renal angiomyolipomas of patients with TSC may mute the malignant potential of these tumors by decreasing Akt activation. MATERIALS AND METHODS Cell Culture and Adenovirus Infection-Tsc2ϩ/ϩ and Tsc2 Ϫ/Ϫ MEFs, generously provided by Dr. D. J. Kwiatkowski (Harvard University), were grown in DMEM with low glucose with 10% fetal bovine serum (22,23). 293 cells were grown in DMEM with high glucose-containing 10% serum. All cell stocks were maintained in the presence of plasmocin and primocin. Tsc2 Ϫ/Ϫ MEFs were infected with adenovirus vector expressing TSC2. This viral vector also expressed green fluorescence protein to detect efficient infection. As a control, an adenovirus vector expressing ␤-galactosidase was used.

MicroRNA-214 Reduces Insulin-like Growth Factor-1 (IGF-1) Receptor Expression and Downstream mTORC1 Signaling in Renal Carcinoma Cells

“…Site-directed Mutagenesis-Four bases in the seed sequence of miR-214 recognition element in the IGF-1R 3ЈUTR-Luc were mutated using QuikChange II site-directed mutagenesis kit as described (36). The mutation was verified by sequencing the DNA.…”

Unrestrained Mammalian Target of Rapamycin Complexes 1 and 2 Increase Expression of Phosphatase and Tensin Homolog Deleted on Chromosome 10 to Regulate Phosphorylation of Akt Kinase

“…ϩ/ϩ and TSC2 Ϫ/Ϫ MEFs were grown as described previously in DMEM with low glucose in the presence of 10% fetal bovine serum (41). 293 cells were grown in DMEM with high glucose in 10% fetal bovine serum.…”

“…Cell Lysis and Immunoblotting-MEFs and 293 cells treated with rapamycin (25 nM) or transfected cells were washed with PBS and harvested in radioimmune precipitation assay buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM Na 3 VO 4 , 1 mM PMSF, 0.1% protease inhibitor mixture, and 1% Nonidet P-40) (41,44,45). The crude cell extracts were centrifuged at 12,000 ϫ g at 4°C for 30 min.…”

“…A second level of negative regulation on PI 3-kinase/Akt signaling is achieved by down-regulation of PDGF receptor-␤ in TSC2-null murine embryonic fibroblasts (MEFs) via an unknown mechanism (39,40). We have recently reported that in TSC2-null MEFs, the level of PTEN is significantly elevated, which also contributes to decreased phosphorylation of Akt (41). The signal transduction mechanism by which PTEN is up-regulated in TSC2-disrupted cells is not known.…”

Regulation of PI 3-K, PTEN, p53, and mTOR in Malignant and Benign Tumors Deficient in Tuberin

“…26 The complete loss of tuberin expression that we recently reported in the malignant renal tumors of Eker rats indicates that the loss of both TSC-2 alleles is an important event in malignant tumor development. 9 This is supported by our observation that in the benign AML tissues, only partial loss of tuberin expression is observed.…”