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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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.