Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR
Hongbing Zhang, Gregor Cicchetti, Hiroaki Onda, Henry B. Koon, Kirsten Asrican, Natalia Bajraszewski, Francisca Vazquez, Christopher L. Carpenter, David J. Kwiatkowski
Hongbing Zhang, Gregor Cicchetti, Hiroaki Onda, Henry B. Koon, Kirsten Asrican, Natalia Bajraszewski, Francisca Vazquez, Christopher L. Carpenter, David J. Kwiatkowski
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Article Oncology

Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR

Abstract

Tuberous sclerosis (TSC) is a familial tumor syndrome due to mutations in TSC1 or TSC2, in which progression to malignancy is rare. Primary Tsc2–/– murine embryo fibroblast cultures display early senescence with overexpression of p21CIP1/WAF1 that is rescued by loss of TP53. Tsc2–/–TP53–/– cells, as well as tumors from Tsc2+/– mice, display an mTOR-activation signature with constitutive activation of S6K, which is reverted by treatment with rapamycin. Rapamycin also reverts a growth advantage of Tsc2–/–TP53–/– cells. Tsc1/Tsc2 does not bind directly to mTOR, however, nor does it directly influence mTOR kinase activity or cellular phosphatase activity. There is a marked reduction in Akt activation in Tsc2–/–TP53–/– and Tsc1–/– cells in response to serum and PDGF, along with a reduction in cell ruffling. PDGFRα and PDGFRβ expression is markedly reduced in both the cell lines and Tsc mouse renal cystadenomas, and ectopic expression of PDGFRβ in Tsc2-null cells restores Akt phosphorylation in response to serum, PDGF, EGF, and insulin. This activation of mTOR along with downregulation of PDGFR PI3K-Akt signaling in cells lacking Tsc1 or Tsc2 may explain why these genes are rarely involved in human cancer. This is in contrast to PTEN, which is a negative upstream regulator of this pathway.

Authors

Hongbing Zhang, Gregor Cicchetti, Hiroaki Onda, Henry B. Koon, Kirsten Asrican, Natalia Bajraszewski, Francisca Vazquez, Christopher L. Carpenter, David J. Kwiatkowski

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Effects of inhibitors on S6K/S6 phosphorylation and growth of TP53–/–Tsc...
Effects of inhibitors on S6K/S6 phosphorylation and growth of TP53–/–Tsc2–/– and control TP53–/– MEFs. (a) Immunoblot analysis of a serum-starved (2 days) then stimulated TP53–/–Tsc2+/+ cell line (top) and a serum-starved TP53–/–Tsc2–/– cell line (bottom). Cells were treated with 10 μM LY294002, 0.1 μM wortmannin, 10 nM rapamycin, 0.1 μM calyculin A, 5 μM TPCK, 10 μM U0126, or 20 μM PD98059 for 30 minutes. (b) Growth effects in two TP53–/–Tsc2–/– (open squares) and two TP53–/–Tsc2+/+ (filled triangles) cell lines of treatment with rapamycin. Each symbol reflects a consecutive day in culture. Rapamycin selectively reduces the growth of the TP53–/–Tsc2–/– cell lines in 0% serum. (c) Immunoblot analysis of effects of 1- and 2-butanol treatment on S6K/S6 phosphorylation in TP53–/–Tsc2–/– and control TP53–/– cell lines. 1- or 2-butanol (0.3%) were applied to the cell lines for 30 minutes, and the cells were serum stimulated for 5 minutes. (d) Immunoblot analysis of effects of AA deprivation and stimulation on S6K/S6 phosphorylation in two TP53–/–Tsc2–/– cell lines. All treatments were for 2 hours. Note that with either the EBSS or HBSS buffers, AAs are required to maintain pS6K and pS6 phosphorylation.