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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Reduced PI3K-Akt signaling in Tsc2–/–TP53–/– and Tsc2–/– cells. (a) Immu...
Reduced PI3K-Akt signaling in Tsc2–/–TP53–/– and Tsc2–/– cells. (a) Immunoblot analysis of cell extracts showing reduced pAkt in Tsc2–/–TP53–/– in comparison to control TP53–/– cells at all time points after 10% serum addition. pAkt S473, pAkt (Ser473). (b) Immunoblot analysis showing reduced pAKT in serum-starved Tsc1–/– and Tsc2–/–TP53–/– cells with and without treatment with calyculin A. (c) Autoradiography demonstrates reduced PI3K activity in Tsc2–/–TP53–/– cells in comparison with TP53–/– cells, in response to PDGF. p-tyrosine, phosphotyrosine; PIP, phosphoinositide phosphate. (d) Ruffling activity, indicated by arrowheads, in response to PDGF was reduced in Tsc2–/–TP53–/– cells in comparison to control TP53–/– cells or a revertant TSC2-expressing cell line. Error bars (n = 3) depict the SD. (e) YPH-Akt translocation is reduced in Tsc2–/–TP53–/– cells in comparison to control TP53–/– cells or a revertant TSC2-expressing cell line. PDGF stimulation leads to uniform YFP staining of the plasma membrane in Tsc2+/+ and revertant cells, but not in Tsc2–/– cells. The staining intensity in cross-sections of the same cells is shown in the graphs at right. AU, arbitrary units.