Role of the F-box protein Skp2 in lymphomagenesis

Abstract

The F-box protein Skp2 (S-phase kinase-associated protein 2) positively regulates the G(1)-S transition by controlling the stability of several G(1) regulators, such as the cell cycle inhibitor p27. We show here that Skp2 expression correlates directly with grade of malignancy and inversely with p27 levels in human lymphomas. To directly evaluate the potential of Skp2 to deregulate growth in vivo, we generated transgenic mice expressing Skp2 targeted to the T-lymphoid lineage as well as double transgenic mice coexpressing Skp2 and activated N-Ras. A strong cooperative effect between these two transgenes induced T cell lymphomas with shorter latency and higher penetrance, leading to significantly decreased survival when compared with control and single transgenic animals. Furthermore, lymphomas of Nras single transgenic animals often expressed higher levels of endogenous Skp2 than tumors of double transgenic mice. This study provides evidence of a role for an F-box protein in oncogenesis and establishes SKP2 as a protooncogene causally involved in the pathogenesis of lymphomas.

Figure 1

Skp2 expression correlates directly with grade of malignancy and inversely with p27 levels in human lymphomas. (A) Characterization of novel mAbs to Skp2 by straight immunoblot. Lanes 1, 4, 7, and 10: 25 μg of extract from human transformed T lymphocytes (Jurkat); lanes 2, 5, 8, and 11: 25 μg of extract from mouse diploid T lymphocytes; lanes 3, 6, 9, and 12: 10 ng recombinant histidine-tagged Skp2. Each panel corresponds to a different membrane immunoblotted with the indicated affinity-purified anti-Skp2 mAb. Only clone 2C2B12 recognized both human and mouse Skp2 (which migrates faster than human Skp2). (B) Immunohistochemistry of two representative lymphomas, with the use of a mix of the four novel affinity-purified anti-Skp2 mAbs (Upper) or an anti-p27 mAb (Lower). (Left) A high-grade lymphoma (DLCL). (Right) A low-grade lymphoma (SLL). All photos were taken at the same microscope magnification (×40). Small p27-positive cells in DLCL represent normal lymphocytes infiltrating the tumor, which, in contrast, has larger mostly negative cells. (C) The expression of Skp2 and p27 was analyzed by immunohistochemistry in 30 cases of high-grade (DLCL) and 28 cases of low-grade (SLL) human lymphomas. Tumors were divided into positive (>25% of positive cells) and negative (<25% of positive cells). (D) Scatter diagram showing the distribution of Skp2 and p27 fractions in DLCL and SLL.

Figure 2

Characterization of CD4-SKP2 transgenic mice. (A) Expression of Skp2 and other cell cycle regulatory proteins in a control nontransgenic mouse (Ctr, lanes 1–3) and in CD4-SKP2 transgenic mice, line 2 (S-2, lanes 4–6) and line 16 (S-16, lanes 7–9). Extracts (30 μg of protein) from thymocytes (T, lanes 1, 4, and 7), unstimulated lymphocytes from lymph nodes (L, lanes 2, 5, and 8), and lymphocytes stimulated for 48 h with phorbol 12-myristate 13-acetate + ionomycin (SL, lanes 3, 6, and 9) were immunoblotted with the antibodies to the indicated proteins. Skp2 mAb (clone 2C2B12) recognized both murine endogenous Skp2 (endog. Skp2) and human exogenous Skp2 (exog. Skp2). Human Skp2 migrates more slowly than murine Skp2 (see also Fig. 1A). The fourth panel from the top was immunoblotted with a p27 phospho-Thr-187 site-specific antibody (11) (indicated as P-p27). (B) Association of human Skp2 with endogenous murine Cul1 and p27. Extracts from thymocytes of a control nontransgenic mouse (Ctr, lane 1) and a CD4-SKP2 transgenic mouse, line 16 (S-16, lane 2) were first immunoprecipitated with a rabbit anti-Skp2 antibody that specifically recognizes human Skp2 (7) and then immunoblotted with antibodies to Skp2, Cul1, and p27. (C) p27 ubiquitinating activity in thymic extracts (30 μg of protein) from control (Ctr, lanes 1 and 2) and transgenic (S-16, lanes 3 and 4) mice. To assay for p27 ubiquitination, extracts from thymocytes were incubated for 90 min (lanes 1 and 3) or 120 min (lanes 2 and 4) in the presence of cyclin E/Cdk2. ³⁵S-methionine-labeled in vitro-translated p27 was used as a substrate as described (7, 11). The bracket on the left side of the panels marks a ladder of bands greater than 27,000 corresponding to polyubiquitinated p27.

Figure 3

Skp2 cooperates with N-Ras to induce T cell lymphomas in mice. (A) Percentage of survival in progeny of transgenic crosses of CD4-SKP2 mice (lines 2 and 16) and TMTV-Nras mice (line A). Nontransgenic control mice and single transgenic mice are indicated as Ctr, Skp2, and N-Ras, respectively; double transgenic mice are indicated as Skp2 + N-Ras. The percentage of survivors is given against the age in days. No significant difference in survival was observed when lines 2 and 16 were analyzed independently (not shown). (B) Immunoblotting of tissue extracts (30 μg of protein) from control thymi (Ctr) and tumors developed in TMTV-Nras mice (R) or in double transgenic mice (S + R). Numbers represent different animals from which tissues were obtained. Extracts were immunoblotted with the antibodies to the indicated proteins. The asterisk in the E2F-1 panel indicates a nonspecific band.

Figure 4

Increased sensitivity to a suboptimal mitogenic stimulus in thymocytes from young double transgenic mice. [³H]thymidine incorporation in thymocytes from control nontransgenic mice (Ctr) and mice expressing Skp2 and N-Ras transgenic proteins. Thymocytes were treated with Con A (A) or with Con A and IL-2 (B) for 72 h. [³H]thymidine was added to the medium during the last 24 h. The results are shown as the mean of [³H]thymidine incorporation obtained in several independent experiments with seven nontransgenic mice, three CD4-SKP2 mice, five TMTV-Nras mice, and eight double transgenic mice.