Unsupervised Natural Experience Rapidly Alters Invariant Object Representation in Visual Cortex

cells with constructs encoding both FBXW7 and HA-ubiquitin, and found that ubiquitination of mTORwas restored by exogenousFBXW7 expression (Fig. 2E). Thus, ubiquitination of mTOR is largely, if not exclusively, mediated by binding to FBXW7. As FBXW7 and PTEN both affect signaling through mTOR, we examined the genetic status of both genes in a panel of 53 breast cancer cell lines (11). Quantitative TaqMan real-time polymerase chain reaction (PCR) assays of the number of copies of FBXW7 and PTEN genes in each of the cell lines were in good concordance with data found by bacterial artificial chromosome (BAC) comparative genomic hybridization (CGH) microarray (see table S1). Most of the breast cancer cell lines that exhibited loss of a single copy of FBXW7 (23 out of 53, Fig. 3A) did not show corresponding loss of PTEN. In contrast, of the 14 lines that showed loss of a single copy of PTEN (Fig. 3A), only one had also lost a copy ofFBXW7, which suggested that FBXW7 and PTEN show some functional redundancy in tumor development. Similar results were obtained by examination of the copy number status of genomic regions containing FBXW7 and PTEN genes in three independent human primary breast cancer sets for which BACCGHmicroarray data were available (12–14). From a total of 450 tumor and cell line DNA samples shown in Fig. 3, A to D, only 4 had lost a copies of the regions containing both genes, a result that is unlikely to be a consequence of random genetic alterations (P = 4.9 × 10). We also considered the possibility that other somatic changes such as point mutations or genesilencing events could affect the results. TheFBXW7 gene continued to be expressed in all 25 breast cancer cell lines examined (fig. S8), which indicated that no gene silencing had occurred, although very low levels were found in five cell lines [lanes 10, 13, 14, 16, and 20 (fig. S8)]. All of these lines had lost one copy of the FBXW7 gene except one (SUM149PT, lane 16), in which a point mutation was detected (table S1). The PTEN gene was found to be silent in two cell lines (fig. S8, lanes 11 and 12), and both had lost one copy of the PTEN gene. Three mutations in PTEN were found (fig. S8 and table S1). Thus, gene silencing (for example, by promoter methylation) or point mutations in FBXW7 and PTEN are relatively rare mechanisms of inactivation of these genes, in comparison with single-copy deletions. These data are further compatible with the identification of both genes as haplo-insufficient tumor suppressors (3, 15, 16). Because deletion or mutation of FBXW7 in human breast cancer cells leads to increased levels of mTOR, we tested the possibility that cells harboring these deletions may show increased sensitivity to themTOR inhibitor rapamycin.We treated twobreast cancer cell lines,SUM149PTcells (homozygous FBXW7 mutations) and MDA-MB453 cells (wild-type FBXW7) with rapamycin and counted numbers of viable cells. SUM149PTcells proved to be very sensitive to this treatment [median inhibitory concentration (IC50) < 200 nM], whereas MDA-MB453 cells were relatively resistant (IC50 > 2 mM) (Fig. 4A). In nude mouse xenografts, groups of fivemice were injected with both cell lines, one on each flank, and were treated by intraperitoneal injection with rapamycin over an 11-day period. The SUM149PT cells showed a relative decrease in size followed by stable tumor growth, whereas the MDA-MB453 cells were relatively unaffected by treatment (Fig. 4B). An additional set of 10 breast cancer cell lines was treated with rapamycin at concentrations of 200 and 400 nM. Cells with deletion or mutation of FBXW7 (HBL100, 600MPE, SUM149PT, HCC3153, and HCC1143) or PTEN (HCC1937 and HCC3153) showed significant sensitivity to killing by rapamycin, although the magnitude of the effect varied (17) (Fig. 4C). To establish a direct link between loss of FBXW7 and rapamycin sensitivity, we down-regulated expression levels of FBXW7 using short hairpin RNA (shRNA) (18) in the rapamycin-resistantMDA-MB453 cells, which resulted in an increase in sensitivity to this drug [IC50< 0.8mM (Fig. 4D)]. Our findings implicate FBXW7 in an evolutionarily conserved pathway that controls regulation of mTOR protein levels. Because FBXW7 is a haploinsufficient tumor suppressor that undergoes heterozygous loss in a substantial proportion of human tumors, the data suggest new approaches to reducemTOR levels in cancers by the use of drugs thatmay reactivate the remaining copy of FBXW7 in a similarway that nutlins (small-moleculeMDM2antagonists) have been shown to activate wild-type copiesof p53 inhuman tumors (19). LossofFBXW7 may also be a useful biomarker for sensitivity of human tumors to inhibitors of themTORpathway. References and Notes 1. M. Welcker, B. E. Clurman, Nat. Rev. Cancer 8, 83 (2008). 2. S. Akhoondi et al., Cancer Res. 67, 9006 (2007). 3. J. H. Mao et al., Nature 432, 775 (2004). 4. H. Rajagopalan et al., Nature 428, 77 (2004). 5. M. Yada et al., EMBO J. 23, 2116 (2004). 6. M. Welcker et al., Proc. Natl. Acad. Sci. U.S.A. 101, 9085 (2004). 7. Z. Kemp et al., Cancer Res. 65, 11361 (2005). 8. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr; and X, any amino acid. 9. G. Wu et al., Mol. Cell. Biol. 21, 7403 (2001). 10. H. Strohmaier et al., Nature 413, 316 (2001). 11. R. M. Neve et al., Cancer Cell 10, 515 (2006). 12. J. Climent et al., Cancer Res. 67, 818 (2007). 13. K. Chin et al., Cancer Cell 10, 529 (2006). 14. J. Fridlyand et al., BMC Cancer 6, 96 (2006). 15. J. H. Mao et al., Oncogene 22, 8379 (2003). 16. A. Di Cristofano, B. Pesce, C. Cordon-Cardo, P. P. Pandolfi, Nat. Genet. 19, 348 (1998). 17. L. S. Steelman et al., Oncogene 27, 4086 (2008). 18. M. Welcker, A. Orian, J. E. Grim, R. N. Eisenman, B. E. Clurman, Curr. Biol. 14, 1852 (2004). 19. J. K. Buolamwini et al., Curr. Cancer Drug Targets 5, 57 (2005). 20. We thank B. Vogelstein for providing us with the HCT116 WT, HCT116 FBXW7, DLD1 wild-type, and DLD1 FBXW7 cell lines; K.I. Nakayama for providing Fbxw7 knockout mice and vectors (HA-FBXW7 and HA-FBXW7DF); and O. Tetsu for vector encoding HA-ubiquitin. These studies were supported by NCI grant U01 CA84244 and the U.S. Department of Energy (DE-FG02-03ER63630) to A.B. , the University of California at San Francisco Research-Evaluation Allocation Committee (REAC) to J.-H.M. A.B. acknowledges support from the Barbara Bass Bakar Chair of Cancer Genetics.