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

García-Beccaria M, Martínez P, Méndez-Pertuz M, Martínez S, Blanco-Aparicio C, Cañamero M, Mulero F, Ambrogio C, Flores JM, Megias D, Barbacid M, Pastor J, Blasco MA - EMBO Mol Med (2015)

Bottom Line: This is accompanied by induction of telomeric DNA damage, apoptosis, decreased proliferation, and G2 arrest.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.

View Article: PubMed Central - PubMed

Affiliation: Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.

No MeSH data available.


Related in: MedlinePlus

Trf1 downregulation in K-RasG12V-transformed lung cells leads to a decreased tumor growth and decreased metastatic potential in allograft and xenograft modelsA–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.D Representative images of the subcutaneous tumors.E Trf1 expression levels measured by qPCR in the injected cell line and in the generated subcutaneous tumors.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.I Representative images of aberrant giant nuclei and anaphase bridges in the Trf1-downregulated subcutaneous tumors compared to the normal nuclei of control tumors.J TRF1 immunofluorescence shows the downregulation of Trf1 in lung tumors of the mice intravenously injected with control and Trf1-downregulated cells.K Tumor area measured in the lungs of the mice intravenously injected with control and Trf1-downregulated cells.L Representative images of the lungs colonized by control and Trf1-downregulated cells, respectively.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.P Trf1 expression levels measured by qPCR in the A549 cell line infected either with sh-scrambled or sh-Trf1.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.
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fig05: Trf1 downregulation in K-RasG12V-transformed lung cells leads to a decreased tumor growth and decreased metastatic potential in allograft and xenograft modelsA–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.D Representative images of the subcutaneous tumors.E Trf1 expression levels measured by qPCR in the injected cell line and in the generated subcutaneous tumors.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.I Representative images of aberrant giant nuclei and anaphase bridges in the Trf1-downregulated subcutaneous tumors compared to the normal nuclei of control tumors.J TRF1 immunofluorescence shows the downregulation of Trf1 in lung tumors of the mice intravenously injected with control and Trf1-downregulated cells.K Tumor area measured in the lungs of the mice intravenously injected with control and Trf1-downregulated cells.L Representative images of the lungs colonized by control and Trf1-downregulated cells, respectively.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.P Trf1 expression levels measured by qPCR in the A549 cell line infected either with sh-scrambled or sh-Trf1.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.

Mentions: To validate these results using an independent strategy to inhibit TRF1, as well as to assess the effect of TRF1 abrogation in already established K-Ras-induced lung tumors, we downregulated Trf1 expression by using shRNA technology in three K-RasΔ/G12Vgeop53−/− mouse cancer cell lines derived from three independent mouse lung carcinoma lesions and assessed the effects on tumor growth using two independent allograft experiments. First, to address the effect of TRF1 inhibition in tumor growth in vivo, we subcutaneously injected 50,000 lung carcinoma cells into immunodeficient mice and followed tumor onset and growth. Trf1 downregulation resulted in a marked delay in tumor onset as well as in a significantly decreased tumor growth (Fig5A–D). Three weeks after injection, mice were sacrificed and the tumors were histologically analyzed. We confirmed that Trf1 downregulation was maintained during in vivo tumor development (Fig5E). Trf1-downregulated tumors showed decreased proliferation and increased DNA damage as well as increased apoptosis compared to controls (Fig5F–H). Again, Trf1-downregulated tumors presented a high proportion of aberrant nuclei and anaphase bridges (Fig5I). Next, to study the effect of TRF1 inhibition in the metastatic potential of established lung cancer cells induced by K-Ras expression, we intravenously injected 150,000 K-RasΔ/LG12Vgeop53−/− lung cells into immunodeficient mice. Tail vein injection of tumor cells results in lung metastasis (Elkin & Vlodavsky, 2001). Three weeks after injection, the mice were sacrificed and lungs were subjected to full histopathology analysis. Again, we confirmed that Trf1 downregulation was maintained in the generated lung metastasis (Fig5J). Importantly, Trf1 downregulation resulted in smaller lung metastasis (Fig5K and I), coincidental with increased DNA damage, decreased proliferation, and increased apoptosis compared to the controls (Fig5M–O). These results indicate that even a partial decrease in TRF1 levels of approximately 50% in tumors very significantly impairs lung tumor growth and lung metastasis, arguing that putative small molecule inhibitors of TRF1 could be effective.


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

García-Beccaria M, Martínez P, Méndez-Pertuz M, Martínez S, Blanco-Aparicio C, Cañamero M, Mulero F, Ambrogio C, Flores JM, Megias D, Barbacid M, Pastor J, Blasco MA - EMBO Mol Med (2015)

Trf1 downregulation in K-RasG12V-transformed lung cells leads to a decreased tumor growth and decreased metastatic potential in allograft and xenograft modelsA–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.D Representative images of the subcutaneous tumors.E Trf1 expression levels measured by qPCR in the injected cell line and in the generated subcutaneous tumors.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.I Representative images of aberrant giant nuclei and anaphase bridges in the Trf1-downregulated subcutaneous tumors compared to the normal nuclei of control tumors.J TRF1 immunofluorescence shows the downregulation of Trf1 in lung tumors of the mice intravenously injected with control and Trf1-downregulated cells.K Tumor area measured in the lungs of the mice intravenously injected with control and Trf1-downregulated cells.L Representative images of the lungs colonized by control and Trf1-downregulated cells, respectively.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.P Trf1 expression levels measured by qPCR in the A549 cell line infected either with sh-scrambled or sh-Trf1.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.
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fig05: Trf1 downregulation in K-RasG12V-transformed lung cells leads to a decreased tumor growth and decreased metastatic potential in allograft and xenograft modelsA–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.D Representative images of the subcutaneous tumors.E Trf1 expression levels measured by qPCR in the injected cell line and in the generated subcutaneous tumors.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.I Representative images of aberrant giant nuclei and anaphase bridges in the Trf1-downregulated subcutaneous tumors compared to the normal nuclei of control tumors.J TRF1 immunofluorescence shows the downregulation of Trf1 in lung tumors of the mice intravenously injected with control and Trf1-downregulated cells.K Tumor area measured in the lungs of the mice intravenously injected with control and Trf1-downregulated cells.L Representative images of the lungs colonized by control and Trf1-downregulated cells, respectively.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.P Trf1 expression levels measured by qPCR in the A549 cell line infected either with sh-scrambled or sh-Trf1.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.
Mentions: To validate these results using an independent strategy to inhibit TRF1, as well as to assess the effect of TRF1 abrogation in already established K-Ras-induced lung tumors, we downregulated Trf1 expression by using shRNA technology in three K-RasΔ/G12Vgeop53−/− mouse cancer cell lines derived from three independent mouse lung carcinoma lesions and assessed the effects on tumor growth using two independent allograft experiments. First, to address the effect of TRF1 inhibition in tumor growth in vivo, we subcutaneously injected 50,000 lung carcinoma cells into immunodeficient mice and followed tumor onset and growth. Trf1 downregulation resulted in a marked delay in tumor onset as well as in a significantly decreased tumor growth (Fig5A–D). Three weeks after injection, mice were sacrificed and the tumors were histologically analyzed. We confirmed that Trf1 downregulation was maintained during in vivo tumor development (Fig5E). Trf1-downregulated tumors showed decreased proliferation and increased DNA damage as well as increased apoptosis compared to controls (Fig5F–H). Again, Trf1-downregulated tumors presented a high proportion of aberrant nuclei and anaphase bridges (Fig5I). Next, to study the effect of TRF1 inhibition in the metastatic potential of established lung cancer cells induced by K-Ras expression, we intravenously injected 150,000 K-RasΔ/LG12Vgeop53−/− lung cells into immunodeficient mice. Tail vein injection of tumor cells results in lung metastasis (Elkin & Vlodavsky, 2001). Three weeks after injection, the mice were sacrificed and lungs were subjected to full histopathology analysis. Again, we confirmed that Trf1 downregulation was maintained in the generated lung metastasis (Fig5J). Importantly, Trf1 downregulation resulted in smaller lung metastasis (Fig5K and I), coincidental with increased DNA damage, decreased proliferation, and increased apoptosis compared to the controls (Fig5M–O). These results indicate that even a partial decrease in TRF1 levels of approximately 50% in tumors very significantly impairs lung tumor growth and lung metastasis, arguing that putative small molecule inhibitors of TRF1 could be effective.

Bottom Line: This is accompanied by induction of telomeric DNA damage, apoptosis, decreased proliferation, and G2 arrest.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.

View Article: PubMed Central - PubMed

Affiliation: Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.

No MeSH data available.


Related in: MedlinePlus