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. 2015 Jul;7(7):930-49.
doi: 10.15252/emmm.201404497.

Therapeutic inhibition of TRF1 impairs the growth of p53-deficient K-RasG12V-induced lung cancer by induction of telomeric DNA damage

María García-Beccaria  1 , Paula Martínez  1 , Marinela Méndez-Pertuz  1 , Sonia Martínez  2 , Carmen Blanco-Aparicio  2 , Marta Cañamero  3 , Francisca Mulero  4 , Chiara Ambrogio  5 , Juana M Flores  6 , Diego Megias  7 , Mariano Barbacid  4 , Joaquín Pastor  2 , Maria A Blasco  8
Affiliations

Therapeutic inhibition of TRF1 impairs the growth of p53-deficient K-RasG12V-induced lung cancer by induction of telomeric DNA damage

María García-Beccaria et al. EMBO Mol Med. 2015 Jul.

Abstract

Telomeres are considered anti-cancer targets, as telomere maintenance above a minimum length is necessary for cancer growth. Telomerase abrogation in cancer-prone mouse models, however, only decreased tumor growth after several mouse generations when telomeres reach a critically short length, and this effect was lost upon p53 mutation. Here, we address whether induction of telomere uncapping by inhibition of the TRF1 shelterin protein can effectively block cancer growth independently of telomere length. We show that genetic Trf1 ablation impairs the growth of p53-null K-Ras(G12V)-induced lung carcinomas and increases mouse survival independently of telomere length. This is accompanied by induction of telomeric DNA damage, apoptosis, decreased proliferation, and G2 arrest. Long-term whole-body Trf1 deletion in adult mice did not impact on mouse survival and viability, although some mice showed a moderately decreased cellularity in bone marrow and blood. Importantly, inhibition of TRF1 binding to telomeres by small molecules blocks the growth of already established lung carcinomas without affecting mouse survival or tissue function. Thus, induction of acute telomere uncapping emerges as a potential new therapeutic target for lung cancer.

Keywords: TRF1; cancer; drug development; shelterin; telomeres.

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Figures

Figure 1
Figure 1
Efficient oncogenic K-RasG12V expression and Trf1 depletion in lung lesions

  1. Genetic model. Trf1lox and K-RasLSLGV12 alleles are depicted before and after Cre-mediated excision.

  2. In vivo imaging schedule. Eight- to ten-week-old mice were intratracheally infected with adeno-Cre, mice were analyzed every 2 weeks by computerized tomography (CT), and 22 weeks post-infection, a positron emission tomography (PET) was performed. Mice were sacrificed 24 weeks post-infection for further histological analysis.

  3. TRF1 immunofluorescence of the lungs. Notice the absence and presence of TRF1 signal in the carcinomas and surrounding healthy tissue of Trf1Δ/Δ mice, respectively.

  4. Analysis of Trf1 excision by PCR. Notice the completed excision in carcinomas of Trf1lox/lox lungs.

  5. Detection of β-galactosidase activity in the lungs as a surrogate marker of oncogenic K-RasG12V expression.

Figure 2
Figure 2
Trf1 deficiency impairs K-Ras-mediated lung cancer development

  1. Tumor growth curve of Trf1+/+ K-Ras+/G12V p53+/+ and Trf1Δ/Δ K-Ras+/G12V p53+/+ measured by computed tomography (CT).

  2. Quantification of the number and size of Trf1+/+ K-Ras+/G12V p53+/+ and Trf1Δ/Δ K-Ras+/G12V p53+/+ carcinomas at death point.

  3. Percentage of tumors that have deleted Trf1 quantified by TRF1 immunofluorescence after mice had been sacrificed. Post-mortem analysis of Trf1 deletion in each tumor revealed that none of the Trf1lox/lox K-Ras+/G12V p53+/+ ones had excised Trf1.

  4. Tumor growth curve and tumor growth slope of Trf1+/+ K-Ras+/G12V p53−/− and Trf1Δ/Δ K-Ras+/G12V p53−/− measured by CT.

  5. Tumor maximal section of Trf1+/+ K-Ras+/G12V p53−/− and Trf1Δ/Δ K-Ras+/G12V p53−/− lungs measured by histological analysis before death point by CT.

  6. Maximum 18F-FDG-glucose uptake by Trf1+/+ K-Ras+/G12V p53−/− and Trf1Δ/Δ K-Ras+/G12V p53−/− tumors 22 weeks after infection by positron emission tomography (PET).

  7. Representative PET-CT image of Trf1+/+ K-Ras+/G12V p53−/− and Trf1Δ/Δ K-Ras+/G12V p53−/− lungs.

  8. Survival curve of Trf1+/+ K-Ras+/G12V p53−/− and Trf1Δ/Δ K-Ras+/G12V p53−/− mice.

Data information: Error bars represent standard error. t-test, chi-squared (B) test, or log-rank (Mantel–Cox; H) test was used to assess statistical significance. The number of mice and the number of tumors are indicated in each case.
Figure 3
Figure 3
Trf1-deficient carcinomas present high amount of telomeric DNA damage and apoptosis

  1. Percentage of cells showing γH2AX foci in carcinomas of the indicated genotypes (left panel). Representative images of γH2AX immunohistochemistry (right panel).

  2. Percentage of cells showing 3 or more γH2AX and RAP1 colocalizing foci (TIFs) (left panel). Representative images of γH2AX and RAP1 double immunofluorescence (right panel). Yellow arrowheads: colocalization of γH2AX and RAP1.

  3. Percentage of active caspase-3 (AC3)-positive cells (left panel). Representative images of AC3 immunohistochemistry (right panel).

Data information: Error bars represent standard error. The number of mice and carcinomas analyzed per genotype is indicated. t-test was used to assess statistical significance.
Figure 4
Figure 4
Trf1 deficiency leads to G2 arrest and mitotic defects

  1. Percentage of Ki67-positive cells in the carcinomas of the indicated genotypes (left panel). Representative images of Ki67 immunohistochemistry (right panel).

  2. Percentage of pH3-positive cells in the carcinomas of the indicated genotypes (left panel). Representative images of pH3 immunohistochemistry (right panel). Red arrowheads: pH3-positive cells.

  3. Percentage of giant nuclei in the carcinomas of the indicated genotype.

  4. Percentage of anaphase bridges out of total anaphases in the carcinomas of the indicated genotypes.

  5. Representative images of giant nuclei, multilobulated nuclei, anaphase bridges, and multipolar mitoses. Red arrowheads indicate the corresponding mitotic aberrations indicated in the image.

Data information: Error bars represent standard error. The number of mice and carcinomas analyzed per genotype is indicated. t-test was used to assess statistical significance.
Figure 5
Figure 5
Trf1 downregulation in K-RasG12V-transformed lung cells leads to a decreased tumor growth and decreased metastatic potential in allograft and xenograft models

  1. A–C The latency (A), volume (B), and weight (C) of subcutaneous tumors generated by control and Trf1-downregulated K-RasG12V-transformed lung cells in athymic mice.

  2. D Representative images of the subcutaneous tumors.

  3. E Trf1 expression levels measured by qPCR in the injected cell line and in the generated subcutaneous tumors.

  4. F–H Number of Ki67-positive (F), number of γH2AX-positive (G), and number of active caspase-3-positive (H) cells per field in the subcutaneous tumors.

  5. I Representative images of aberrant giant nuclei and anaphase bridges in the Trf1-downregulated subcutaneous tumors compared to the normal nuclei of control tumors.

  6. J TRF1 immunofluorescence shows the downregulation of Trf1 in lung tumors of the mice intravenously injected with control and Trf1-downregulated cells.

  7. K Tumor area measured in the lungs of the mice intravenously injected with control and Trf1-downregulated cells.

  8. L Representative images of the lungs colonized by control and Trf1-downregulated cells, respectively.

  9. M–O Number of Ki67-positive (M), number of γH2AX-positive (N), and number of active caspase-3-positive (O) cells per field in the lung tumors.

  10. P Trf1 expression levels measured by qPCR in the A549 cell line infected either with sh-scrambled or sh-Trf1.

  11. Q Growth of A549-derived tumors.

Data information: Error bars represent standard error. The number of mice and tumors analyzed per condition is indicated. t-test was used to assess statistical significance.
Figure 6
Figure 6
Systemic Trf1 depletion in healthy tissues does not compromise tissue function nor organism viability

  1. Trf1lox/lox hUBC-CreERT2 genetic mouse model.

  2. Trf1 expression levels in the indicated tissues of wild-type and Trf1lox/lox hUBC-CreERT2 mice subjected to a tamoxifen-containing diet for 7 weeks.

  3. Representative images of TRF1 immunofluorescence and quantification of the percentage of TRF1-positive cells in skin and intestine sections of wild-type and Trf1lox/lox hUBC-CreERT2 mice subjected to a tamoxifen-containing diet for 7 weeks.

  4. Survival curve of wild-type and Trf1lox/lox hUBC-CreERT2 mice subjected to a tamoxifen-containing diet for 7 weeks.

  5. Quantification of the histological alterations observed in tamoxifen-treated Trf1lox/lox hUBC-CreERT2 mice and 4 months after tamoxifen retrieval compared to their wild-type counterparts.

  6. Quantification of blood cell populations in wild-type and Trf1lox/lox hUBC-CreERT2 mice subjected to a tamoxifen-containing diet for 7 weeks and after 3 weeks and 4 months of tamoxifen retrieval.

  7. Trf1 expression levels in blood, intestine, skin, and bone marrow of Trf1lox/lox hUBC-CreERT2 mice subjected to a tamoxifen-containing diet either for 7 weeks or for 6 months and after 3 weeks tamoxifen retrieval compared to wild-type mice.

Data information: Error bars represent standard error. The number of mice analyzed per genotype is indicated in each case. t-test was used to assess statistical significance. TMX, tamoxifen.
Figure 7
Figure 7
Efficient chemical inhibition of TRF1 telomere binding by compounds ETP-47228 and ETP-47037 in mouse lung adenocarcinoma-derived cells

  1. Quantification of TRF1 levels by immunofluorescence in lung tumor-derived cell line treated with DMSO, with 10 μM ETP-47228 (24 h), and with 10 μM ETP-47037 (48 h). Representative images are shown to the right.

  2. Quantification of γH2AX levels by immunofluorescence in lung tumor-derived cell line treated with DMSO, with ETP-47228 (24 h), and with ETP-47037 (48 h). Representative images are shown to the right.

  3. Quantification of telomere-induced foci (TIFs) by double immunofluorescence with anti-RAP1 and anti-γH2AX antibodies. Representative images are shown to the right. White arrowheads: colocalization of γH2AX and RAP1.

  4. Effect of different ETP-47228 and ETP-47037 concentrations during 24 h on proliferation in lung tumor-derived cell line relative to the growth of DMSO-treated cells.

  5. Tumor growth quantification in allograft model ETP-47037 or with ETP-47228.

Data information: The data represent the mean values of two to three independent experiments (A–D). Error bars represent standard errors. t-test was used to assess statistical significance.
Figure 8
Figure 8
“In vivo” treatment with ETP-47037 compound blocks the progression of lung carcinoma

  1. Schematic representation of the ETP-47037 treatment protocol. Mice with already developed lung carcinomas were subjected to computerized tomography (CT) measurements before the start of the treatment. ETP-47037 was given at a dose of 75 mg/kg body weight by oral gavage 8 days out of the ten that the experiment lasted as indicated. At the end of the treatment, a CT was performed for quantification of tumor area and mice were sacrificed for further histological and molecular analysis. Control mice were similarly treated with vehicle.

  2. Quantification of tumor growth relative to initial tumor size. Representative CT images are shown to the right. The white arrowhead indicates a tumor within a highly inflammatory region.

  3. Quantification of TRF1 levels by immunofluorescence in intestine and lung tumors samples of mice treated with vehicle or with ETP-47037 for 10 days. Representative images are shown to the right (n = 4).

  4. Number of cells showing γH2AX foci in intestine and lung tumors samples of mice treated with vehicle or with ETP-47037 for 10 days. Representative images are shown to the right (n = 4).

Data information: The data represent the mean values obtained from three mice in each group. Error bars represent standard errors. t-test was used to assess statistical significance.
Figure 9
Figure 9
“In vivo” treatment with ETP-47037 compound does not compromise tissue viability

  1. A–C Quantification of the number of (A) Ki67-, (B) pan-nuclear p-H3 pattern-, and (C) foci p-H3 pattern-positive cells in untreated and ETP-470037-treated lung carcinomas. The data represent the mean values obtained for three mice in each group. Error bars represent standard errors.

  2. D Representative Ki67 and p-H3 images.

  3. E Representative H&E images of intestine samples corresponding to untreated and ETP-47037-treated animals. High-magnification images are shown to the right indicating the presence of normal mitosis, giant multinucleated and aberrant mitotic figures.

  4. F Representative H&E images of bone marrow and skin samples corresponding to untreated and ETP-47037-treated animals. High-magnification images are shown indicating the presence of necrosis, hemosiderosis, multinucleated cells, and giant nuclei. Bone marrow showed moderated aplasia.

  5. G Telomere length in untreated and ETP-47037-treated lung tumor samples. Representative images are shown to the right.

Data information: t-test was used to assess statistical significance.
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