Frap, FKBP12 rapamycin-associated protein, is a candidate gene for the plasmacytoma resistance locus Pctr2 and can act as a tumor suppressor gene

Abstract

Susceptibility to mouse plasmacytomagenesis is a complex genetic trait controlled by several Pctr loci (Pctr1, Pctr2, etc). Congenic strain analysis narrowed the genetic interval surrounding the Pctr2 locus, and genes identified in the interval were sequenced from susceptible BALB/c and resistant DBA/2 mice. Frap (FKBP12 rapamycin-associated protein, mTOR, RAFT) was the only gene differing in amino acid sequence between alleles that correlated with strain sensitivity to tumor development. The in vitro kinase activity of the BALB/c FRAP allele was lower than the DBA/2 allele; phosphorylation of p53 and PHAS1/4EBP1 (properties of heat and acid stability/eukaryotic initiation factor 4E-binding protein) and autophosphorylation of FRAP were less efficient with the BALB/c allele. FRAP also suppressed transformation of NIH 3T3 cells by ras, with DBA/2 FRAP being more efficient than BALB/c FRAP. Rapamycin, a specific inhibitor of FRAP, did not inhibit growth of plasmacytoma cell lines. These studies identify Frap as a candidate tumor suppressor gene, in contrast to many reports that have focused on its prooncogenic properties. Frap may be similar to Tgfb and E2f in exerting both positive and negative growth-regulatory signals, depending on the timing, pathway, or tumor system involved. The failure of rapamycin to inhibit plasma cell tumor growth suggests that FRAP antagonists may not be appropriate for the treatment of plasma cell tumors. Pctr2 joins Pctr1 in possessing alleles that modify susceptibility to plasmacytomagenesis by encoding differences in efficiency of function (efficiency alleles), rather than all-or-none, gain-of-function, or loss-of-function alleles. By analogy, human cancer may also result from the combined effects of several inefficient alleles.

Fig. 1.

Definition of the Pctr2 interval by congenic strain and sequence analysis. The blue bar represents a portion of band 4E on mouse Chr 4. The intervals of several C.D2 congenic strains are shown under the chromosome with the associated DBA/2 alleles represented by the horizontal black bars. The tumor phenotype is indicated as “S” for susceptible and “R” for resistant. The red vertical lines represent the proximal and distal boundaries of the Pctr2 interval. With respect to gene order, Xmv8 is more centromeric and D4Mit225 is telomeric. Results for the C.D2-Fv1B strain are shown in Fig. 2. The other strains are described in ref. .

Fig. 2.

A congenic strain further defines the genetic interval surrounding the Pctr2 locus. The Xmv8–Xmv44 interval was ruled out as a potential location for Pctr2. These studies, coupled with earlier backcross studies, confine the Pctr2 interval to a region of distal mouse Chr 4 surrounding the microsatellite marker D4Mit310.

Fig. 3.

Evolutionary conservation of FRAP. The polymorphism found between BALB/c and DBA/2 mice at residue 628 represents a rare allele in the BALB/c strain. Arginine has been conserved at this residue over the course of 1.2 billion years.

Fig. 4.

In vitro kinase activity of FRAP. Anti-FLAG immunoprecipitates of HEK293 cells transiently transfected with plasmid encoding FLAG-tagged BALB/c FRAP or DBA/2 FRAP (Right) were tested in an in vitro kinase reaction with PHAS1 and p53 added as exogenous substrate (Left). DBA/2 FRAP kinase was more active in phosphorylating FRAP, itself, PHAS1, and p53. Kinase assays were repeated three times with similar results.

Fig. 5.

Inhibition of ras-mediated focus formation by FRAP. NIH 3T3 cells were cotransfected with BALB/c and DBA/2 variants of FRAP along with v-ras. The percentages of residual foci formed relative to the foci detected on the plates cotransfected with pcDNA vector control and v-ras are shown. Transfection assays were repeated three times with similar results. DBA FRAP was more efficient at suppressing ras transformation than BALB/c FRAP.

Fig. 6.

Resistance of plasmacytoma cell lines to rapamycin. OVCAR4 and PC3 cells were used as control cell lines that are known to respond to rapamycin treatment with an increase in the number of cells arresting in the G₁ phase of the cell cycle. Treatment of plasmacytoma cell lines with rapamycin did not induce an increase in cells in G₁. Mutations were not detected in the region surrounding the rapamycin-binding FRB domain of Frap in the plasmacytoma cell lines examined (see Methods).