Short telomeres limit tumor progression in vivo by inducing senescence

Abstract

Telomere maintenance is critical for cancer progression. To examine mechanisms of tumor suppression induced by short telomeres, we crossed mice deficient for the RNA component of telomerase, mTR(-/-), with Emu-myc transgenic mice, an established model of Burkitt's lymphoma. Short telomeres suppressed tumor formation in Emu-myc transgenic animals. Expression of Bcl2 blocked apoptosis in tumor cells, but surprisingly, mice with short telomeres were still resistant to tumor formation. Staining for markers of cellular senescence showed that pretumor cells induced senescence in response to short telomeres. Loss of p53 abrogated the short telomere response. This study provides in vivo evidence for the existence of a p53-mediated senescence mechanism in response to short telomeres that suppresses tumorigenesis.

Figure 1

Short telomeres decrease penetrence of tumor formation and initiate genomic instability. A) Kaplan-Meier survival analysis showing lymphoma onset in Myc mTR+/+ (black, n= 31), Myc mTR -/- G1 (blue, n= 18) and Myc mTR -/- G5/6 (red, n=25) cohorts. B) Quantitative fluorescencein situ hybridization (Q-FISH) of tumor cell metaphase spreads from Myc mTR +/+ (top), Myc mTR -/- G1 (middle), and Myc mTR -/- G6 (bottom). Ten metaphases each from three separate lymphomas are represented for each genotype. C) Representative Q-FISH (top) and spectral karyotypes (bottom) for Myc mTR +/+ (left) and Myc mTR -/- G6 (right) tumors. Arrow heads indicate end-to-end chromosome fusions and non-reciprocal translocations in Q-FISH and SKY images respectively. D) Quantitation of chromosome abnormalities. The number of chromosome end-to-end fusions (red bars) and non-reciprocal translocations (blue bars) per metaphase are shown (10 metaphases per tumor n=3). Bars indicate standard error.

Figure 2

Short telomeres suppress lymphoma formation despite abrogation of the apoptotic program. A) Experimental bone marrow transplant scheme. Bone marrow from donors of defined genotypes was infected with Bcl-2 expressing murine stem cell virus prior to transplantation into lethally irradiated wildtype recipient animals. Iconography reproduced by kind permission of New Science Press fromImmunity (DeFranco et al., 2007).B) Kaplan-Meier survival analysis of Myc mTR +/+ (black, n=8), Myc mTR -/-G1 (blue, n=7), and Myc mTR -/- G5/6 (red, n=3(G5), n=5 (G6)) cohorts. C) TUNEL analysis of tumor masses from Bcl-2; Myc mTR+/+, Bcl-2; Myc mTR-/- G5, and Bcl-2 Myc mTR-/- G6. Bars indicate standard error.

Figure 3

Short telomeres block tumor formation in a p53 dependent manner. A) Intergenerational breeding scheme. Vertical bars represent telomere lengths. B) Kaplan-Meier survival analysis of Myc iG7 mTR +/- p53 +/+ (black dashed, n=11) Myc iG7 mTR -/- p53 +/+ (red dashed, n=20), Myc iG7 mTR +/- p53 +/-(black solid, n=7), and Myc iG7 mTR -/- p53 +/- (red, solid n=9) cohorts. Myc p53+/- animals invariably develop Myc p53-/- tumors at around 40 days. Short telomeres promote chromosomal abnormalities in p53 deficient lymphomas. C) Q-FISH analysis of metaphase spreads from Myc mTR +/- p53 -/- (top) and Myc mTR -/-p53 -/- (bottom) lymphomas. Ten metaphases each from three separate lymphomas are represented for each genotype. D) Quantitation of chromosome abnormalities. The number of chromosome end-to-end fusions (red bars) and non-reciprocal translocations (blue bars) per metaphase are shown. (10 metaphases per tumor n=3). Bars indicate standard error.

Figure 4

A) Loss of apoptotic induction in tumor cells derived from Myc mTR-/- G5/6 mice. Six hours after 0 or 5 Gy γ-IR cells were stained with Annexin-V FLUOS (FL-1) and propidium iodide (FL-3) and processed by flow cytometry. Representative tumors from Myc mTR +/+ (top) and Myc mTR -/- G6 (bottom) are shown. Sensitivity to γ-IR is shown by loss of viable cells (lower left quadrant) after irradiation. B) Summary of the percent of viable cells, as described in A, for all tumors analyzed. C) Immunoblot of non-irradiated or irradiated (5 Gy γ-IR) tumor cell lysates for p53, Arf, and Actin. Representative tumors from Myc mTR +/+ (left panel) and Myc mTR -/- G5/6 (right panel). Normal p53 induction shown in lanes 1-2, and 5-6, overexpression of mutant form in lanes 3-4, 7-8, and 11-12. Complete loss of p53 protein is shown in lanes 9-10, and 13-14. Arf expression is seen only in samples with suspected p53 mutations (Eischen et al., 1999). D) Radio-resistant DNA synthesis in lymphomas with p53 mutations. Cells were treated with 5 Gy γ-IR after 1 hour BrdU was added to culture medium then six hours post irradiation cells were fixed and stained with anti-BrdU antibody and subjected to FACS analysis (FL-3 = 7-AAD signal and FL-1 = anti-BrdU FLUOS) the boxed area indicates cells in S-phase.

Figure 5

Micro-lymphomas from Bcl-2; Myc mTR-/- G5/6 animals are senescent. A) Mitotic Indices for Bcl-2; Myc mTR+/+, Bcl-2; Myc mTR-/- G5, and Bcl-2; Myc mTR-/- G6. Bars indicate standard error. B) Histological analysis of Bcl-2; Myc mTR+/+ and Bcl-2; Myc mTR-/- G6 lymphomas. Top) H&E stain showing complete effacement of cervical lymph node in Bcl-2; Myc mTR +/+ and small encapsulated micro-lymphoma surrounded by salivary glands in Bcl-2; Myc mTR-/- G6. Senescence associated β-gal activity, p16^INK4a and p15^INK4b immuno-stain specifically associated with Bcl-2; mTR -/- G6 micro-lymphomas. Scale bar represents 250 μm for low power hematoxylin and eosin (H&E) stained images (top panels) and 100 μm for all other images.

Figure 6

Tumor suppressor signaling downstream of short telomeres and myc. The myc oncogene negatively regulates its effects on cell proliferation by inducing both Arf and Bim tumor suppressors. Arf activity results in stabilization of the p53 protein, whereas Bim is directly transcribed by myc and induces apoptosis by inhibiting Bcl-2. Activation of both Arf and Bim is required for myc induced apoptosis. Apoptotic signaling can also be disrupted by over-expression of the downstream anti-apoptotic Bcl-2 oncogene. The short telomere response signals through both p16 and p53 tumor suppressor pathways, which carry out senescence and/or apoptotic tumor suppressor functions. The p53 tumor suppressor lies at the crossroads of senescence and apoptosis and is required for both programs.