Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR
Hongbing Zhang, … , Christopher L. Carpenter, David J. Kwiatkowski
Hongbing Zhang, … , Christopher L. Carpenter, David J. Kwiatkowski
Published October 15, 2003
Citation Information: J Clin Invest. 2003;112(8):1223-1233. https://doi.org/10.1172/JCI17222.
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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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Figure 6

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PDGFR is reduced in Tsc2–/–TP53–/– and Tsc1–/– cells. (a) Immunoblot ana...
PDGFR is reduced in Tsc2–/–TP53–/– and Tsc1–/– cells. (a) Immunoblot analysis of cell extracts demonstrates that both PDGFRβ and PDGFRα levels are reduced in both Tsc1-null and Tsc2-null cell lines compared with controls, while expression of insulin receptor α (IRα) is similar in these cells. Expression of PDGFRβ is reduced in tumor (T) extracts compared with normal kidney (NK) from both Tsc2+/– and Tsc1+/– mice. (b) Left top five rows, immunoblot analysis of cell extracts showing reduced amount of PDGFRβ in the Tsc2–/–TP53–/– cell line compared with the TP53–/– control. Left bottom three rows, analysis of PDGFRβ immunoprecipitations (IPs) showing reduced amount of PDGFRβ, pPDGFRβ, and bound PI3K p85 subunit in the Tsc2–/–TP53–/– cell extracts. Right, immunoblot showing expression of PDGFRβ is restored in a TSC2-expressing revertant TP53–/– cell line (pEF6/TSC2). (c) Autoradiogram showing levels of PDGFRβ in a pulse-chase experiment with S35-methionine labeling in TP53–/– cells. Levels of PDGFRβ are decreased at the 0 time point in both of the Tsc2–/– cell lines compared with controls, but levels decline similarly during the chase. (d) BrdU incorporation experiment for serum-starved Tsc2-null, control, and revertant TP53–/– cells in response to treatment for 12 hours with 25 ng/ml PDGF with or without 25 μM AG17 or 50 ng/ml EGF and 10 μM BrdU. Left, serum starvation for 1 day; right, for 3 days. Similar results were obtained using cell counting.