Specific tumor suppressor function for E2F2 in Myc-induced T cell lymphomagenesis

Abstract

Deregulation of the Myc pathway and deregulation of the Rb pathway are two of the most common abnormalities in human malignancies. Recent in vitro experiments suggest a complex cross-regulatory relationship between Myc and Rb that is mediated through the control of E2F. To evaluate the functional connection between Myc and E2Fs in vivo, we used a bitransgenic mouse model of Myc-induced T cell lymphomagenesis and analyzed tumor progression in mice deficient for E2f1, E2f2, or E2f3. Whereas the targeted inactivation of E2f1 or E2f3 had no significant effect on tumor progression, loss of E2f2 accelerated lymphomagenesis. Interestingly, loss of a single copy of E2f2 also accelerated tumorigenesis, albeit to a lesser extent, suggesting a haploinsufficient function for this locus. The combined ablation of E2f1 or E2f3, along with E2f2, did not further accelerate tumorigenesis. Myc-overexpressing T cells were more resistant to apoptosis in the absence of E2f2, and the reintroduction of E2F2 into these tumor cells resulted in an increase of apoptosis and inhibition of tumorigenesis. These results identify the E2f2 locus as a tumor suppressor through its ability to modulate apoptosis.

MYC is often amplified in human cancers, and mouse models of cancer have demonstrated a causal role for MYC overexpression in hematopoietic, mammary, and other cancer types (1–3). Deregulation of the Rb/E2F gene networks also represents common events in cancer (4). Like Myc, E2F can positively and negatively regulate the expression of hundreds of targets whose gene products are involved in a wide spectrum of biological processes, with a bias for genes that control cell cycle, apoptosis, and differentiation (5–10).

Based on amino acid sequence analysis and structure–function studies in vitro, E2F family members can be artificially grouped into activator (E2F1–3) and repressor (E2F4–8) subclasses (11). Because of the intense interest in E2Fs as major regulators of the cell cycle, individual E2F family members have also been extensively studied in vivo by gene-targeting approaches in mice. E2f1^−/− mice are viable and suffer from impaired thymocyte apoptosis, defective negative selection, and testicular atrophy. E2f2^−/− mice are also viable and have a mild increase in hematopoietic and autoreactive T cells. Much later in life, a portion of E2f1^−/− and E2f2^−/− mice develop hematopoietic malignancies (12–15). These mutant phenotypes might reflect the particular bias for the expression of E2f1 and E2f2 in hematopoietic tissues. Although disruption of the E2f3 gene in a mixed genetic background yields viable mice, its disruption in pure strains results in embryonic lethality at around embryonic day 12.5 (G.L., unpublished observation). Surprisingly, embryos deficient for each of these E2Fs have no apparent defect in cellular proliferation, raising the possibility of functional redundancy among members of the activator subclass of E2Fs. There also appears to be functional redundancy among members of the repressor subclass, because disruption of E2f4, E2f5, or E2f6 in mice has little consequence on the proliferative capacity of cells, but the combined disruption of E2f4 and E2f5 or E2f4 and E2f6 results in the inappropriate proliferation and expression of target genes in response to specific antiproliferative signals (16, 17).

Recent observations in cell culture systems indicate extensive cross-regulation between the action of Myc and E2Fs in coordinating the control of cellular proliferation. Myc action can be funneled by a number of concerted mechanisms to control E2F activity (5, 18), including through the regulation of their expression (7). Although Myc can certainly influence E2F activities, it is also true that Myc can be influenced by E2Fs. How this complex cross-regulatory relationship between Myc and E2F is effectively orchestrated in vivo remains poorly understood. In this study, we used a mouse model of Myc-induced T cell lymphomagenesis along with mice deficient for each of the E2F activators to directly examine the connection between Myc action and these E2Fs in vivo.

Results

E2f2 Locus Harbors Tumor Suppressor Function.

To examine the connection between Myc and E2F transcription factors in vivo, we used a conditional bitransgenic mouse model of MYC-induced T cell lymphomagenesis (19). In this system, expression of EμSR-tTA mediates the transcription of the Teto-MYC transgene in B and T cells and results in the development of predominantly immature T cell lymphomas. These tumors invade the spleen, lymphatics, bone marrow, and blood and eventually lead to the death of mice by 4 months of age.

To explore the possibility that E2f1, E2f2, and E2f3 play a role in MYC-induced lymphomagenesis, we first determined whether these E2Fs are expressed in hematopoietic cell lineages. As assessed by real-time RT-PCR assays, E2f1, E2f2, and E2f3 are expressed in all of the main hematopoietic organs, including bone marrow, spleen, thymus, and lymph nodes (Fig. 1A). Most other organs tested expressed significantly lower levels of E2f1 and E2f3 and essentially little or no E2f2. It is not clear whether the particular high expression of E2f2 in hematopoietic organs is a reflection of the high levels of E2f2 transcripts in these compartments or is just a reflection of the low basal levels of E2f2 in other tissues. We also examined the expression of these three E2fs in T cell tumors that developed in EμSR-tTA;Teto-MYC mice. This analysis revealed a significant reduction in E2f2 expression in most tumors tested but little effect on the expression of E2f1, E2f3a, and E2f3b (Fig. 1B and data not shown). The basis for the reduction in E2f2 expression remains to be elucidated, but we speculated that it might be related to its potential role in the MYC-induced tumorigenic process.

Fig. 1.

Loss of E2f2 accelerates Myc-induced T cell lymphomagenesis. (A) Real-time RT-PCR analysis of the expression of E2f1, E2f2, E2f3a, and E2f3b in normal mouse tissues (b.m., bone marrow; l.n., lymph node). The relative values were determined by comparing the expression of the indicated mRNAs to the expression of GAPDH. The data are shown as induction (n-fold) of gene expression in tissues relative to expression in the liver that was set to one. One representative example of three independent experiments is shown. Error bars represent mean ± SD. Values that exceeded the y-axis scale are shown on the top of interrupted bars. (B) Real-time RT-PCR analysis of E2f1, E2f2, E2f3a, and E2f3b expression in normal mouse thymocytes (controls) and MYC-induced T cell lymphomas (tumors). (C) Kaplan–Meier survival curves (time from birth to death) of the EμSR-tTA (tetracycline controlled transactivator);Teto-MYC (+/+) and EμSR-tTA;Teto-MYC cohorts of mice containing heterozygous (+/−) or homozygous (−/−) mutations of indicated E2fs. Survival curve for EμSR-tTA;Teto-MYC is shown by solid black line. Curves for the cohorts of mice containing deletion of E2f2 allele are shown by red lines, whereas curves for the cohorts of mice that did not involve deletion of E2f2 are shown in blue. Dashed lines and solid lines indicate heterozygous and homozygous deletion of E2fs, respectively. The number of mice used in each cohort is indicated by n. Student's t test was used for statistical analyses as described in Materials and Methods, and P values are shown. (D) Kaplan–Meier survival curves of the EμSR-tTA;Teto-MYC ([+/+;+/+]; black line), EμSR-tTA;Teto-MYC;Teto-Cre;E2f1^−/−;E2f3^LoxP/LoxP ([−/−;LoxP/LoxP]; red line), EμSR-tTA;Teto-MYC;E2f1^−/−;E2f2^−/− ([−/−;−/−]; red line), or EμSR-tTA;Teto-MYC;Teto-Cre;E2f2^−/−;E2f3^LoxP/LoxP ([−/−;LoxP/LoxP]; red line) cohorts of mice. (E) Kaplan–Meier survival curves of the EμSR-tTA;Teto-MYC ([+/+;+/+;+/+]; black line) and EμSR-tTA;Teto-MYC;Teto-Cre;E2f1^−/−;E2f2^−/−;E2f3^LoxP/LoxP ([+/+;+/+;LoxP/LoxP]; red line). (F) Conditional deletion of the E2f3^LoxP allele in T cell lymphomas derived from EμSR-tTA;Teto-MYC;Teto-Cre;E2f3^LoxP/LoxP or EμSR-tTA;Teto-MYC;Teto-Cre;E2f3^+/LoxP mice as analyzed by Southern blotting using an E2f3-specific probe. Nondeleted E2f3^LoxP/LoxP genomic DNA served as a control. The position of bands corresponding to E2f3 knockout (−), E2f3^LoxP (LoxP), and E2f3 wild-type (+/−) alleles is indicated.

To rigorously assess the physiological role of E2F activators in MYC-induced lymphomagenesis, we initially generated cohorts of EμSR-tTA;Teto-MYC mice that lacked either one or both alleles of E2f1 or E2f2. These mice were monitored for tumor formation over a period of 1 year, as described (19). Time of death was noted and plotted on Kaplan–Meier survival graphs. As shown previously, EμSR-tTA;Teto-MYC mice succumbed to tumors by 110 days of age with a median survival (MS) of 83 days [confidence interval (C.I.) 78–85 days; n = 109]. The inactivation of E2f1 had no significant effect on the MS of these tumor mice (Fig. 1C). Strikingly, the inactivation of E2f2 significantly accelerated tumor onset and progression, resulting in their death at a median age of 67 days (C.I., 65–70 days; n = 128; P < 0.001; Fig. 1C). Interestingly, loss of even one copy of E2f2 significantly accelerated disease progression when compared with the control wild-type cohort (E2f2^+/+, P < 0.001), albeit to a lesser extent than when both alleles were deleted. Tumors derived from E2f2 heterozygous animals retained the wild-type allele, as assessed by Southern blot analysis [supporting information (SI) Fig. 5 and data not shown], suggesting that small changes in the levels of E2F2 protein could have a substantial impact in tumorigenesis. Characterization of tumors by cell surface marker expression confirmed that control and E2f2-deficient tumors were positive for CD3 and TCRβ and negative for the B cell marker B220 or myeloid marker CD11b (data not shown), indicating tumors were of T cell origin. In each case, tumors consisted of either CD4/CD8 double-positive cells or CD4 single-positive cells (SI Fig. 6). Together, these results formally demonstrate a haploinsufficient tumor suppressor function for the E2f2 locus in T cell lymphomagenesis.

Germ-line inactivation of E2f3 in an FVB strain background results in embryonic lethality at around embryonic day 12.5, precluding tumor studies with these mice (G.L., unpublished observations). To circumvent the problem of embryonic lethality, we analyzed tumor development in EμSR-tTA;Teto-MYC mice containing the Teto-Cre transgene and a conditional allele of E2f3 (E2f3^LoxP). In these mice, tTA driven from the EμSR-tTA transgene results in the expression of both the Teto-MYC and Teto-Cre transgenes. Thus, MYC expression and Cre-mediated ablation of E2f3 can be achieved within the same subset of hematopoietic cells. As shown in Fig. 1C, there was no significant difference in the MS time between the EμSR-tTA;Teto-MYC;E2f3^LoxP/LoxP and EμSR-tTA;Teto-MYC;Teto-Cre;E2f3^LoxP/LoxP groups of mice (P = 0.375). Southern blot analysis and PCR-based genotyping confirmed the complete ablation of E2f3 in tumors arising in EμSR-tTA;Teto-MYC;Teto-Cre; E2f3^LoxP/LoxP mice (Fig. 1F and data not shown), suggesting that loss of E2f3 function was not selected against during tumorigenesis. Moreover, Cre expression itself did not appear to affect tumor outcome, because cohorts of mice containing the Teto-Cre transgene had identical MS times as those lacking the Teto-Cre transgene (data not shown). These results strongly suggest that E2f3 does not significantly contribute to MYC-induced lymphomagenesis. Because E2f3 was conditionally deleted in hematopoietic lineages, as opposed to the global deletion of E2f1 or E2f2, we cannot rule out the formal possibility that inactivation of E2f3 in the germ line would have a different phenotypic consequence on T cell lymphomagenesis than observed here.

E2f1 and E2f3 Are Dispensable for MYC-Induced Lymphomagenesis.

Functional compensation among E2F family members, as demonstrated in fibroblasts cultured in vitro (20), may explain the lack of an effect on tumor outcome imparted by the loss of either E2f1 or E2f3. We therefore analyzed disease progression in mice deleted for both E2f1 and E2f3. Surprisingly, EμSR-tTA;Teto-MYC;E2f1^−/−;E2f3^LoxP/LoxP and EμSR-tTA;Teto-MYC;Teto-Cre;E2f1^−/−;E2f3^LoxP/LoxP mice developed T cell lymphomas with similar kinetics as wild-type mice (Fig. 1D). Southern blot analysis of tumor DNA confirmed the complete deletion of E2f3 in tumors derived from mice expressing Cre (SI Fig. 7A and data not shown). From these in vivo studies, we conclude that E2f1 and E2f3 do not play a measurable role in MYC-induced lymphomagenesis.

E2f2's Tumor Suppressor Function Is Independent of E2f1 and E2f3.

The tumor studies described above demonstrate specificity among E2fs in the manifestation of tumor outcome; however, because of the incredible functional plasticity among E2F family members (17), it is difficult to ascertain the molecular basis for this specificity. Although no significant change in the expression of other E2F family members was observed in tumors deficient for E2f2 (data not shown), it is possible that E2F1 and/or E2F3 action might have been rerouted in E2f2-deficient cell to perform functions not normally performed in cells containing all three activator E2Fs. These “acquired functions” could be responsible for diminishing or accentuating the manifestation of tumor outcome resulting from the loss of E2f2. To test this possibility, we created cohorts of EμSR-tTA;Teto-MYC;E2f2^−/− mice that were also deficient for either E2f1, E2f3, or both. As shown in Fig. 1D, EμSR-tTA;Teto-MYC;E2f1^−/−;E2f2^−/− and EμSR-tTA;Teto-MYC;Teto-Cre;E2f2^−/−;E2f3^LoxP/LoxP cohorts had a similar MS time as EμSR-tTA;Teto-MYC;E2f2^−/− mice (66, 67, and 67 days, respectively). Surprisingly, the simultaneous inactivation of all three E2F activators in EμSR-tTA;Teto-MYC;Teto-Cre;E2f1^−/−;E2f2^−/−;E2f3^LoxP/LoxP mice resulted in a similar progression of disease as in E2f2-deficient mice (MS time of 65 and 67 days, respectively; Fig. 1E). Once again, Southern blot analysis confirmed the complete deletion of E2f3^LoxP/LoxP in the subset of tumors expressing Cre (SI Fig. 7 B and C). Based on these data, we conclude that E2f2's tumor suppressor role in T cell lymphomagenesis is independent of E2f1 and E2f3.

Cell-Autonomous Tumor Suppressor Function of E2F2.

The global inactivation of E2f2 precluded us from making any conclusions relating to where the critical action of E2F2 for suppressing lymphomagenesis might reside. To examine whether loss of E2f2 accelerated the tumorigenic process in a cell-autonomous manner, we used an adoptive transfer strategy to introduce E2f2-deficient fetal liver cells into wild-type irradiated animals. To this end, lethally irradiated recipient FVB mice were injected with fetal liver cells isolated from EμSR-tTA;Teto-MYC and EμSR-tTA;Teto-MYC;E2f2^−/− 15.5-day-old embryos. Injected mice were then monitored for tumor formation over a period of 1 year (25 mice per genetic group; see SI Fig. 8A). Approximately 60% of mice (14/25) from the group that received EμSR-tTA;Teto-MYC cells developed T cell lymphomas, as confirmed by FACS-based immunophenotyping and histological analysis (SI Fig. 8B and data not shown) and succumbed to tumors with a MS time of 160 days. The other 40% of mice remained healthy during the 300-day observation period. In contrast, most mice (22/25) that received EμSR-tTA;Teto-MYC;E2f2^−/− fetal liver cells developed T cell lymphomas and had a MS time of 110 days (SI Fig. 8A). Lymphomas that lacked E2f2 were immunophenotypically indistinguishable from those that had E2f2. Statistical comparison between these two groups showed a significant difference in their MS (P = 0.019). These studies demonstrate that the tumor suppressor function of E2f2 resides within the hematopoietic compartment and is likely cell-autonomous.

Tumor Analysis in E2f2-Deficient Mice.

Loss of E2f2 has been shown to lead to autoimmune disease in older mice due to enhanced TCR-stimulated proliferation and the accumulation of autoreactive effector/memory T lymphocytes (14). We therefore explored whether E2f2's tumor suppressor function described above might be causally related to a T cell differentiation defect, which could be divided into four substages depending on the expression of CD25 and CD44 markers: DN1 (CD44⁺CD25⁻), DN2 (CD44⁺CD25⁺), DN3 (CD44⁻CD25⁺), and DN4 (CD44⁻CD25⁻) (21). To this end, we analyzed the expression of CD44, CD25, CD4, and CD8 in thymocytes derived from 21-day-old wild-type E2f2^−/−, EμSR-tTA;Teto-MYC, and EμSR-tTA;Teto-MYC;E2f2^−/− mice. Loss of E2f2 in either a nontumor or tumor setting had no appreciable effect on the distribution of DN1–DN4 cells or the proportion of CD4 or CD8 single or CD4–CD8 double-positive cells (Fig. 2A and data not shown). We did observe, however, that in contrast to the monoclonal nature of tumors derived from EμSR-tTA;Teto-MYC mice (19), the vast majority of tumors in the E2f2^+/− and E2f2^−/− background were oligoclonal, with homozygously deleted mice having tumors composed of up to five different clones that expressed different TCR receptor isoforms (Fig. 2B).

Fig. 2.

Decreased apoptosis in mice deficient for E2f2 during tumorigenesis. (A) Development of double-negative thymocytes in 21-day-old E2f2^+/+ (nontransgenic; white bars), E2f2^−/− (nontransgenic; black bars), EμSR-tTA;Teto-MYC;E2f2^+/+ (EμSR-tTA;Teto-MYC; white bars), EμSR-tTA;Teto-MYC;E2f2^−/− (EμSR-tTA;Teto-MYC; black bars) as assessed by FACS. Staining with anti-CD4 and -CD8 antibody was used to determine CD4⁻CD8⁻ double-negative population within the live lymphoid gate. Double-negative populations were subsequently analyzed for CD44 and CD25 surface expression. Data are presented as an average percentage ± SD for DN1 (CD44⁺), DN2 (CD44⁺CD25⁺), DN3 (CD44⁻CD25⁺), and DN4 (CD44⁻CD25⁻). (B) Graphic representation of tumor clonality in EμSR-tTA;Teto-MYC mice of the following genotypes: E2f2^+/+, E2f2^+/−, and E2f2^−/−, as determined by analysis of different tissues using a panel of monoclonal antibodies recognizing different TCR Vβ chains. (C) BrdU incorporation and apoptosis assays of EμSR-tTA;Teto-MYC;E2f2^+/+ (white bars) or EμSR-tTA;Teto-MYC;E2f2^−/− (black bars) mice at final stages of disease as determined by FACS using anti-BrdU or anti-Annexin V antibodies. The number of mice used for each cohort is indicated by n. Student's t test was used for statistical analyses, and P values are shown.

In view of the well established role of E2Fs in the control of cellular proliferation and apoptosis, we investigated whether changes in these two processes may represent the basis for the tumor suppressor function of E2F2. To this end, we measured proliferation and apoptotic indices in control and E2f2-deficient littermate animals at early and late stages of tumor development. Analysis of precancerous (21 days of age) or terminally sick mice failed to reveal any substantial difference in the proliferation of control and E2f2-deficient T cells (Fig. 2C and data not shown). In contrast, AnnexinV staining revealed that E2f2-deficient T cells were more resistant to apoptosis than control cells at both early and late stages of tumor development, suggesting that a loss of E2f2 may confer a selective advantage for the expansion of MYC-expressing T cells.

Molecular Characterization of T Cell Lymphomas in E2f2-Deficient Mice.

To identify relevant downstream activities that might be responsible for the observed acceleration of tumorigenesis in E2f2-deficient mice, we analyzed the expression of cell cycle inhibitors that have previously been implicated in cancer biogenesis. First, we performed real-time RT-PCR expression analysis of the MYC-transgene itself and two known MYC-target genes, Ornithine decarboxylase (Odc) and Nucleolin, in thymic tumor masses from terminally sick EμSR-tTA;Teto-MYC and EμSR-tTA;Teto-MYC;E2f2^−/− mice. This analysis confirmed that the expression of the MYC transgene and its two target genes was similarly activated in all tumors examined when compared with T cells from aged-matched normal control mice (Fig. 3A and data not shown).

Fig. 3.

Expression of cell cycle-regulated genes in E2f2-deleted cells. (A) Real-time RT-PCR analysis of MYC, ODC, p19^ARF, p73, p21^Cip1, and p27^Kip1 expression in normal thymocytes (E2f2^+/+), E2f2-deficient thymocytes (E2f2^−/−), tumors derived from EμSR-tTA;Teto-MYC;E2f2^+/+ or EμSR-tTA;Teto-MYC;E2f2^−/− mice, as indicated. Sequences of primers used for tested genes are shown in SI Table 1. (B) Western blot analysis of p27^Kip1 expression in normal thymocytes (N) and EμSR-tTA;Teto-MYC tumors nondeleted or deleted for E2f2 as indicated.

Previous work using the Eμ-MYC model of B cell lymphoma demonstrated that MYC-induced B cell lymphomagenesis requires the inactivation of apoptotic checkpoints, and that this is frequently achieved through disabling the Arf-p53 pathway (22). We therefore assessed the status of p53 in tumors arising in EμSR-tTA;Teto-MYC and EμSR-tTA;Teto-MYC;E2f2^−/− mice. Direct sequencing of p53 cDNA prepared from 10 tumors did not reveal any mutation in its coding sequence (SI Text). We then analyzed the expression of p19^ARF, whose induction had been shown to be associated with the overexpression of MYC (23). As might have been expected, we observed a dramatic induction of p19^ARF expression in all tumors analyzed, but this induction was independent of the status of E2f2 (Fig. 3A). Consistent with the conclusion that the p53 pathway is not differentially impacted by the presence or absence of E2f2, the expression of one of its target genes, p21^Cip1, did not change in response to the loss of E2f2 (Fig. 3A). Because the expression of p73, a proapoptotic member of the p53 family, has been implicated as a downstream target of E2F1 during TCR activation-induced cell death of peripheral T cells (24), we examined its expression in normal and tumor tissues from E2f2-deficient mice. We found that p73 transcripts were significantly decreased in tumor samples, but this decrease did not depend on the status of E2f2 (Fig. 3A). In contrast, the expression of the antiapoptotic version of p73, deltaNp73, was unchanged between normal and tumor samples (SI Fig. 9). These results suggest that E2F2's tumor suppressor function is independent of the p53 apoptotic axis.

The ability of Myc to impact the cell cycle at least partially depends on its ability to down-regulate p27^Kip1 (25), a cyclin-dependent kinase inhibitor known to contribute to the regulation of the cell cycle and to be down-regulated in a number of tumor settings (26, 27). Consistent with a posttranscriptional mechanism of regulation, p27^Kip1 protein levels but not its mRNA levels, were substantially decreased in tumor cells derived from EμSR-tTA;Teto-MYC or EμSR-tTA;Teto-MYC;E2f2^−/− mice (Fig. 3 A and B). Although this down-regulation of p27^Kip1 likely represents an important event in MYC-induced T cell lymphomagenesis, its regulation would appear not to be linked to E2F2's tumor suppressor function.

Reintroduction of E2f2 Activity into E2F2-Deficient Cells Inhibits Tumorigenesis.

To further examine the role of E2f2 in lymphomagenesis, we evaluated T cells isolated from late-stage tumors that were reconstituted with exogenous E2F2. Tumor cells derived from EμSR-tTA;Teto-MYC;E2f2^−/− mice readily adapted to in vitro growth and could be efficiently infected with retroviral expression vectors (Fig. 4 A and B). Flow cytometric analysis in multiple experiments showed that 20–80% of tumor cells infected with a control or E2F2-expressing MSCV-IRES-GFP vector were GFP-positive (Fig. 4B). Subcutaneous injection of these cells resulted in the formation of tumors within 2 weeks. Essentially all of the tumor cells that emerged from the injection of E2F2-transduced cells were GFP-negative (>99% GFP-negative; Fig. 4 B and C). In contrast, tumors that emerged from the injection of control-transduced cells retained the same percentage of GFP-positive cells as observed before the injection. These experiments reveal a profound bias against the formation of tumors originating from E2F2 overexpressing T cells.

Fig. 4.

Proapoptotic tumor suppressor function of E2f2. (A) Western blot analysis of T cells derived from EμSR-tTA;Teto-MYC;E2f2^−/− tumors infected with MSCV-IRES-EGFP empty vector (con) or MSCV-E2F2-IRES-EGFP (E2F2) using anti-E2f2 antibody. Tubulin served as loading control. (B) FACS analysis of unselected cells infected with the indicated retroviruses before injection into nude mice (Upper). Representative examples of FACS analysis of individual tumors that developed in nude mice (Lower). The percentage of GFP-positive and -negative cells is indicated within the FACS diagrams. (C) Analysis of tumors that developed in mice injected with EμSR-tTA;Teto-MYC;E2f2^−/− tumor T cells that were infected either with control or E2F2 retroviruses. The data are presented as the average ratio of percent of GFP-positive cells in each individual tumor relative to the percent of GFP-positive cells before injection. n indicates a number of tumors analyzed for each group. (D) In vitro proliferation assay of unselected cells infected with the indicated retroviral constructs. Cells were plated at a concentration of 0.2 × 10⁶ per ml (day 0) and counted every 24 h for 7 days. (E) Cells were treated as in D, and the percentage of GFP-positive cells was determined by FACS. The infection efficiency at day 0 (28% for the MSCV-IRES-EGFP vector control and 39% for MSCV-E2F2-IRES-EGFP) was set to 100%. The values obtained for percentage of GFP-positive cells at each time point were plotted relative to the percentage at day 0. (F and G) The percentage of BrdU- and Annexin V-positive cells infected with the indicated retroviruses was measured 2 and 4 days after infection. For D–G, representative examples from three independent experiments are shown.

Alternatively, control and E2F2-transduced cells were plated on tissue culture plates, and their growth was monitored over the course of ≈8 days. In these assays, we could measure a moderate but consistent reduction in the proliferation of cells infected with E2F2-expressing vectors (Fig. 4D). Importantly, comparison of GFP-positive and -negative cells by FACS analysis revealed a profound decrease over time in the percentage of GFP-positive cells transduced with E2F2-vectors (Fig. 4E). In contrast, GFP-positive and -negative cells in control-treated samples proliferated equally well. BrdU incorporation assays indicated that the number of GFP-positive cells entering S phase was not influenced by the over-expression of E2F2 (Fig. 4F and data not shown). AnnexinV assays, however, revealed that the ratio of GFP-positive/-negative apoptotic cells was markedly increased in populations transduced with E2F2-expressing vectors but not with control vectors (Fig. 4G). These results suggest a strong bias against the proliferation of cells expressing the E2F2 protein that is based on its ability to potently induce apoptosis.

In parallel experiments, we could show that overexpression of E2F1 and E2F3a in E2f2-deficient cells could also induce apoptosis and preclude the growth and tumorigenicity of tumor cells (SI Fig. 10 A–F and data not shown). These results suggest that using overexpression approaches, any of the three E2F activators can engage apoptotic pathways and thus eliminate tumor cells. We view these results to indicate that gene ablation strategies can reveal functional specificity with greater fidelity than by overexpression strategies.

Discussion

The E2f2 Locus Harbors Tumor Suppressor Function.

Overexpression of the MYC oncogene and inactivation of the RB tumor suppressor pathway are hallmarks of human cancers. Recent in vitro experiments suggest a complex cross-regulatory relationship between Myc and Rb that is mediated through the control of E2F activities. Here, we used a bitransgenic mouse model of MYC-induced T cell lymphomagenesis and mice deficient for E2f1, E2f2, or E2f3 to evaluate the functional relationship between Myc and E2Fs in vivo. These experiments demonstrate a unique tumor suppressor role for E2F2 in T cell lymphomagenesis. Adoptive transfer experiments show that E2f2's tumor suppressor function resides within the hematopoietic compartment and is therefore likely to be cell-autonomous. Loss of even one allele significantly accelerated tumorigenesis, indicating that tumor progression is sensitive to small changes in total E2F2 protein. This raises the possibility that polymorphisms in the genome that result in lower levels of E2F2 protein, directly or indirectly, may place individuals at a higher risk for cancer development.

E2f1 and E2f3 Are Not Required for Lymphomagenesis.

Based on their shared abilities to control cell proliferation and apoptosis, a functional connection between the Myc and E2F pathways has been long speculated. Most recently, work in mouse embryo fibroblasts suggested that two important functions of Myc in the control of proliferation and apoptosis are mediated, at least in part, by E2Fs (28). This work showed that in fibroblasts, the execution of Myc's proliferative arm requires E2f2 and E2f3, and the execution of its apoptotic arm requires E2f1. This bifurcation of Myc's function at the level of E2F suggested that E2f1 could have tumor suppressor function and E2f2 and E2f3 could have oncogenic functions. These predictions were not born out by the in vivo studies of T cell lymphomagenesis presented here. In fact, mice lacking both E2f1 and E2f3 developed T cell lymphomas with similar kinetics as mice containing a full complement of E2Fs, suggesting that E2f1 and E2f3 do not play a measurable role in MYC-induced lymphomagenesis. The observation that T cells devoid of E2f1, E2f2, and E2f3 proliferated and were fully transformed is quite surprising given our previous work showing that fibroblasts deficient for these E2Fs were unable to proliferate in vitro (20). We do not yet know whether this difference between E2f1/2/3-deficient T cells and fibroblasts reflects a tissue-specific requirement for E2Fs or a consequence of MYC overexpression in T cells. Clearly, important differences must exist between fibroblasts and T cells, and a generalized outcome stemming from the action of E2Fs across different cell contexts may be difficult to predict.

The observation that loss of E2f1 had little bearing on MYC-induced T cell lymphomagenesis is also in direct contradiction to a recent study by Baudino et al. (29), where the authors show that loss of E2f1 dramatically delayed MYC-induced B cell lymphomagenesis. These two different tumor outcomes resulting from the inactivation of E2f1 could reflect inherent differences in the biology of B and T cells. Alternatively, differences in the two E2f1-null alleles (12, 13) or in genetic backgrounds (30, 31) housing environments, methods of tumor analysis, and size of genetic cohorts could account for the observed discrepancies.

Tumor Suppressor Role of E2F2 in Apoptosis.

Two observations indicate that the underlying basis for E2f2's tumor suppressor function is in the control of apoptosis rather than in cell proliferation. First, we observed a decreased number of apoptotic tumor cells in E2f2-null mice. Second, reexpression of E2F2 in E2f2-deficient tumor cells resulted in an increase of apoptosis and abrogation of tumor cell expansion, demonstrating that the critical components required to signal and execute apoptosis remain intact in these tumor cells. In contrast, these loss-of-function and overexpression studies failed to reveal any E2f2-dependent differences in the ability of MYC-expressing tumor cells to replicate their DNA.

The role of E2F2 in apoptosis early on during tumorigenesis could be important in limiting the number of cells susceptible to MYC-induced oncogenesis. This hypothesis would predict that loss of E2f2 might increase the tumor-prone T cell population responsive to MYC overexpression and thus facilitate tumor development. Consistent with this notion, the vast majority of E2f2^+/− and E2f2^−/− mice developed tumors that were oligoclonal in nature, with homozygously deleted tumors expressing up to five different TCR receptor isoforms (Fig. 2B). This is in contrast to the typical monoclonal nature of tumors found in E2f2^+/+ mice. These data suggest a role for E2f2 in both the early and late stages of tumor progression.

Specificity of Function Among E2Fs.

The tumor analysis in E2f-deficient mice clearly demonstrates a specific role for E2f2 in T cell lymphomagenesis. The molecular basis for this tumor suppressor function, however, is less clear. On the one hand, a decrease in E2F2 protein levels but not in E2F1 and E2F3 through the targeted inactivation of E2f2 is sufficient to suppress apoptosis and permit tumor progression. On the other hand, overexpression of E2F2 or E2F1 and, to lesser degree, E2F3a, is able to induce apoptosis in MYC-overexpressing tumor cells. Thus, loss-of-function and overexpression studies appear to lead to contradictory results. Because overexpression of E2Fs can potentially compete with and alter the binding of other E2Fs to target promoters and/or cofactors, we believe that gene ablation approaches are more adept at revealing physiological differences between the function of E2F family members.

What is the underlying reason for the unique tumor suppressor function of E2f2 in MYC-induced lymphomagenesis that is observed in vivo? One possibility is that endogenous levels of E2F2 protein regulate the expression of a specific set of apoptotic-related genes that can be similarly achieved only by other E2Fs when overexpressed at supraphysiological levels. The alternative explanation is that E2f2's tumor suppressor role is not intrinsic to the function of its protein product but rather depends on the magnitude of expression imparted by its locus. In other words, the size of the “total pool” of E2F activity may be the critical variable that determines whether an apoptotic response can be surmounted in face of an oncogenic insult. Although it is possible that the basis for the unique role of E2f2 in MYC-induced lymphomagenesis may stem from quantitative differences in the expression of “activator” E2Fs in T cells, three main reasons argue against this latter possibility. First, if “total” E2F activity was determining tumor suppression, it would be expected that loss of all three E2F activators would accelerate lymphomagenesis further than that observed by the simple loss of E2f2. This prediction was not realized; rather, a deficiency of E2f1/E2f2/E2f3 accelerated lymphomagenesis to the same extent as by loss of E2f2 (Fig. 1 D and E). Second, the absence of “enhanced” tumorigenesis in T cells deficient for all three E2F activators is not simply because E2f1/3 are not expressed to sufficient levels to have a function in T cells. In fact, E2F1 has been shown, as has E2F2, to contribute to normal hematopoiesis in a number of settings. For example, it has been previously shown that in contrast to E2f1^+/−E2f2^−/− or E2f1^−/−E2f2^+/− cells, E2f1^−/−E2f2^−/− bone marrow cells are unable to contribute to development of multiple hematopoietic lineages (33), suggesting that a single allele of E2f1 or E2f2 contribute significantly to this process. Third, in unpublished work from our laboratory, we have observed that loss of E2f2 in tissues that normally express much lower levels of it than in T cells also accelerates tumorigenesis (G.L., unpublished observations). It would thus seem that the tumor suppressor role of E2f2 is not simply due to its abundance in T cells but rather is likely a reflection of its specific function in T cell lymphomagenesis.

Whether E2F2 might also play a role in tumor maintenance remains to be investigated. Further experiments will be necessary to decipher the exact mechanism by which E2F2 exerts its tumor suppressor action in vivo. In summary, these studies reveal specificity among E2F activators in MYC-induced lymphomagenesis, highlighting an apoptotic role for E2f2 in this process.

Materials and Methods

Generation and Maintenance of Mice.

The E2f1^−/−, E2f2^−/−, and Teto-Cre mice were generous gifts from Michael Greenberg (Children's Hospital, Boston, MA), Stuart Orkin (Harvard Medical School, Boston, MA), and Andreas Nagy (Samuel Lunenfeld Research Institute, Toronto, ON, Canada), respectively. The generation of the conditional E2f3 knockout mice (E2f3^LoxP/LoxP) has been described (20). Genotyping of mice was performed by PCR from genomic DNA isolated from mouse tails. All tumor studies were performed with mice bred into FVB (fifth generation). Mice for tumor studies generated using standard genetic procedures were monitored for tumor formation over a period of 1 year, as described (19).

Statistical Analysis.

Kaplan–Meier curves were generated, and MS times with 95% confidence intervals (32) were calculated. Proportional hazards assumptions were confirmed, and the log-rank test was found to be appropriate to compare the survival curves in all cases. Bonferroni adjustments for multiple comparisons were used. Although this is a conservative method of adjustment, the P values found in these data were on the extreme ends, and a less-conservative method would have lead to the same conclusions. In the case where the proportional hazard assumption was not met, such as for the E2f1^−/−E2f3^−/− cohort (Fig. 1D), the survival curves were compared between groups before and after the cross-over of the survival curves.

Abbreviation

MS

median survival.