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Science 321 (5895): 1499-1502

Copyright © 2008 by the American Association for the Advancement of Science

FBXW7 Targets mTOR for Degradation and Cooperates with PTEN in Tumor Suppression

Jian-Hua Mao1*, Il-Jin Kim1*, Di Wu1, Joan Climent1, Hio Chung Kang1, Reyno DelRosario1, and Allan Balmain1,2{dagger}

1 Cancer Research Institute, University of California at San Francisco, 2340 Sutter Street, San Francisco, CA 94143, USA.
2 Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA 94143, USA.

Figure 1 Fig. 1.. Depletion of FBXW7 increases mTOR and p-mTOR levels. (A) The levels of mTOR and p-mTOR, as well as the downstream mTOR target S6-kinase (P-S6K), are up-regulated in Fbxw7–/– MEFs, whereas the levels of Akt and p-Akt (bottom) were not appreciably affected. Two independent preparations were used. (B) Elevation of mTOR in thymus and spleen from Fbxw7+/– mice. Lanes 1 to 3 contain extracts from wild-type, Pten+/–, and Fbxw7+/– mice, respectively. (C) Overexpression of either full-length FBXW7 (HA-FBXW7) or of a dominant-negative form (HA-FBXW7-{Delta}F) in 293T cells, respectively, decreases (lane 2) or increases (lane 3) the mTOR and p-mTOR levels. [View Larger Version of this Image (25K GIF file)]

Figure 2 Fig. 2.. mTOR interacts with FBXW7. (A) Immunoprecipitation (IP) of HA-FBXW7 identifies mTOR as an interacting protein. HA-FBXW7 or FBXW7-{Delta}F was expressed in human 293T cells, followed by immunoprecipitation of the proteins with HA-specific antibodies and immunoblotting (IB) with antibodies against mTOR (top). The reciprocal experiment shows that the region of FBXW7 that interacts is the WD40 domain (middle). (Bottom) The input levels of mTOR and FBXW7 proteins in the lysates. (B) FBXW7 binds the wild-type fragment of mTOR, but binding to the mTOR(delT631) and mTOR (T631G) mutants was dramatically reduced. (C) MCF7 breast cancer cells show increased mTOR levels after treatment with MG-132, but this is not seen in SUM149PT cells, which have no functional FBXW7 gene. (D) HCT116 WT and HCT116 FBXW7–/– cells were transfected with HA-ubiquitin. Immunoprecipitation of mTOR followed by immunoblot analysis of the HA-ubiquitin showed that ubiquitination of mTOR was only seen in the HCT116 WT cells, and not in HCT116 FBXW7–/– cells. The vector lane shows HCT116 FBXW7–/– cells transfected with empty vector construct. (E) Ubiquitination of mTOR is restored by exogenous FBXW7 expression. HCT116 FBXW7–/– cells were transfected with an FBXW7-expressing construct and HA-tagged ubiquitin. Immunoprecipitation of mTOR showed increased ubiquitination compared with controls (fig. S7). [View Larger Version of this Image (24K GIF file)]

Figure 3 Fig. 3.. Genetic interaction between FBXW7 and PTEN in human breast cancers. The green bar indicates loss, the red bar indicates gain, and the black bar indicates no changes. (A) The data from 53 human breast cancer cell lines, ordered in the vertical axis from 1 to 53 (11). The copy number of FBXW7 and PTEN was determined by quantitative PCR with TaqMan. Deletion of FBXW7 rarely occurred in tumors that also show deletion of PTEN (P = 0.014). (B, C, and D) Three independent sets of human primary breast cancers, analyzed by the same BAC CGH microarray platform: (B) 185 human primary breast cancers (12), (C) 145 human primary breast cancers (13), and (D) 67 human primary breast cancers (14). The copy number was determined on the basis of published CGH data (FBXW7 was based on BAC RP11-73G16, PTEN on BAC RP11-380G5). Loss is defined as log2(ratio) < –0.25 and gain as log2(ratio) > 0.25. [View Larger Version of this Image (39K GIF file)]

Figure 4 Fig. 4.. Loss of FBXW7 increases sensitivity to an mTOR inhibitor (rapamycin). (A) The breast cancer SUM149PT cells, which have a homozygous mutation in FBXW7, were killed at a rapamycin concentration of 100 nM, whereas MDA-MB453 cells with wild-type FBXW7 were resistant. (B) Treatment of nude mouse xenografts with rapamycin. The SUM149PT cells showed a relative decrease in size, followed by stable tumor growth, whereas the MDA-MB453 cells were unaffected by treatment. (C) Sensitivity to mTOR rapamycin in a range of breast cancer cell lines. Tumor cells with deletion or mutation of FBXW7 are in group 1, those with deletion or mutation of PTEN are group 2, and cells with wild-type copies of both genes are in group 3. (D) Down-regulation of FBXW7 with specific shRNA in rapamycin-resistant MDA-MB453 cells increases the sensitivity to this treatment. [View Larger Version of this Image (32K GIF file)]

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