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Telomerase inhibition abolishes the tumorigenicity of pediatric ependymoma tumor-initiating cells.

Barszczyk M, Buczkowicz P, Castelo-Branco P, Mack SC, Ramaswamy V, Mangerel J, Agnihotri S, Remke M, Golbourn B, Pajovic S, Elizabeth C, Yu M, Luu B, Morrison A, Adamski J, Nethery-Brokx K, Li XN, Van Meter T, Dirks PB, Rutka JT, Taylor MD, Tabori U, Hawkins C - Acta Neuropathol. (2014)

Bottom Line: Over 60 % of pediatric ependymomas were found to rely on telomerase activity to maintain telomeres, while no ependymomas showed evidence of ALT.Children with telomerase-active tumors had reduced 5-year progression-free survival (29 ± 11 vs 64 ± 18 %; p = 0.03) and overall survival (58 ± 12 vs 83 ± 15 %; p = 0.05) rates compared to those with tumors lacking telomerase activity.Telomerase inhibition represents a promising therapeutic approach for telomerase-active pediatric ependymomas found to characterize high-risk ependymomas.

View Article: PubMed Central - PubMed

Affiliation: The Arthur and Sonia Labatt Brain Tumor Research Centre, The Hospital for Sick Children, Toronto, ON, Canada.

ABSTRACT
Pediatric ependymomas are highly recurrent tumors resistant to conventional chemotherapy. Telomerase, a ribonucleoprotein critical in permitting limitless replication, has been found to be critically important for the maintenance of tumor-initiating cells (TICs). These TICs are chemoresistant, repopulate the tumor from which they are identified, and are drivers of recurrence in numerous cancers. In this study, telomerase enzymatic activity was directly measured and inhibited to assess the therapeutic potential of targeting telomerase. Telomerase repeat amplification protocol (TRAP) (n = 36) and C-circle assay/telomere FISH/ATRX staining (n = 76) were performed on primary ependymomas to determine the prevalence and prognostic potential of telomerase activity or alternative lengthening of telomeres (ALT) as telomere maintenance mechanisms, respectively. Imetelstat, a phase 2 telomerase inhibitor, was used to elucidate the effect of telomerase inhibition on proliferation and tumorigenicity in established cell lines (BXD-1425EPN, R254), a primary TIC line (E520) and xenograft models of pediatric ependymoma. Over 60 % of pediatric ependymomas were found to rely on telomerase activity to maintain telomeres, while no ependymomas showed evidence of ALT. Children with telomerase-active tumors had reduced 5-year progression-free survival (29 ± 11 vs 64 ± 18 %; p = 0.03) and overall survival (58 ± 12 vs 83 ± 15 %; p = 0.05) rates compared to those with tumors lacking telomerase activity. Imetelstat inhibited proliferation and self-renewal by shortening telomeres and inducing senescence in vitro. In vivo, Imetelstat significantly reduced subcutaneous xenograft growth by 40 % (p = 0.03) and completely abolished the tumorigenicity of pediatric ependymoma TICs in an orthotopic xenograft model. Telomerase inhibition represents a promising therapeutic approach for telomerase-active pediatric ependymomas found to characterize high-risk ependymomas.

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Imetelstat-treated cells displayed an activated DNA damage response associated with a progressive increase in senescence. Immunofluorescence showed BXD (a), R254 (b) and E520 (c) cells had increased γH2AX staining compared to untreated and mismatch control cells following 27, 17 and 34 weeks of treatment, respectively. β-galactosidase (β-gal) staining showed a progressive increase in cells undergoing senescence in BXD (d), R254 (e) and E520 (f) cells. All images were taken at 200× magnification and scale bars represent either 50 or 100 μm as indicated. Error bars represent ± SD of the mean. Asterick represents significance at p < 0.05 compared to both untreated and mismatch control
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Fig3: Imetelstat-treated cells displayed an activated DNA damage response associated with a progressive increase in senescence. Immunofluorescence showed BXD (a), R254 (b) and E520 (c) cells had increased γH2AX staining compared to untreated and mismatch control cells following 27, 17 and 34 weeks of treatment, respectively. β-galactosidase (β-gal) staining showed a progressive increase in cells undergoing senescence in BXD (d), R254 (e) and E520 (f) cells. All images were taken at 200× magnification and scale bars represent either 50 or 100 μm as indicated. Error bars represent ± SD of the mean. Asterick represents significance at p < 0.05 compared to both untreated and mismatch control

Mentions: Once telomeres erode to a critically short length, they become dysfunctional and activate a DNA damage response mediated by γH2AX, which ultimately results in senescence [6]. All three cell models treated with Imetelstat showed evidence of γH2AX foci in ~40–60 % of cells by the end of treatment, while almost none of the untreated or mismatch control cells showed evidence of DNA damage (Fig. 3a–c; p < 0.05). BXD, R254 and E520 cells showed a progressive increase in the proportion of Imetelstat-treated cells undergoing senescence, whereby 60, 80 and 30 % of cells, respectively, underwent senescence by the end of treatment (Fig. 3d–f; p < 0.05). Although R254 cells showed a 15 % (p < 0.05) increase in apoptosis in the last 3 weeks of treatment, BXD and E520 cells did not undergo any significant cell death (data not shown).Fig. 3


Telomerase inhibition abolishes the tumorigenicity of pediatric ependymoma tumor-initiating cells.

Barszczyk M, Buczkowicz P, Castelo-Branco P, Mack SC, Ramaswamy V, Mangerel J, Agnihotri S, Remke M, Golbourn B, Pajovic S, Elizabeth C, Yu M, Luu B, Morrison A, Adamski J, Nethery-Brokx K, Li XN, Van Meter T, Dirks PB, Rutka JT, Taylor MD, Tabori U, Hawkins C - Acta Neuropathol. (2014)

Imetelstat-treated cells displayed an activated DNA damage response associated with a progressive increase in senescence. Immunofluorescence showed BXD (a), R254 (b) and E520 (c) cells had increased γH2AX staining compared to untreated and mismatch control cells following 27, 17 and 34 weeks of treatment, respectively. β-galactosidase (β-gal) staining showed a progressive increase in cells undergoing senescence in BXD (d), R254 (e) and E520 (f) cells. All images were taken at 200× magnification and scale bars represent either 50 or 100 μm as indicated. Error bars represent ± SD of the mean. Asterick represents significance at p < 0.05 compared to both untreated and mismatch control
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4286630&req=5

Fig3: Imetelstat-treated cells displayed an activated DNA damage response associated with a progressive increase in senescence. Immunofluorescence showed BXD (a), R254 (b) and E520 (c) cells had increased γH2AX staining compared to untreated and mismatch control cells following 27, 17 and 34 weeks of treatment, respectively. β-galactosidase (β-gal) staining showed a progressive increase in cells undergoing senescence in BXD (d), R254 (e) and E520 (f) cells. All images were taken at 200× magnification and scale bars represent either 50 or 100 μm as indicated. Error bars represent ± SD of the mean. Asterick represents significance at p < 0.05 compared to both untreated and mismatch control
Mentions: Once telomeres erode to a critically short length, they become dysfunctional and activate a DNA damage response mediated by γH2AX, which ultimately results in senescence [6]. All three cell models treated with Imetelstat showed evidence of γH2AX foci in ~40–60 % of cells by the end of treatment, while almost none of the untreated or mismatch control cells showed evidence of DNA damage (Fig. 3a–c; p < 0.05). BXD, R254 and E520 cells showed a progressive increase in the proportion of Imetelstat-treated cells undergoing senescence, whereby 60, 80 and 30 % of cells, respectively, underwent senescence by the end of treatment (Fig. 3d–f; p < 0.05). Although R254 cells showed a 15 % (p < 0.05) increase in apoptosis in the last 3 weeks of treatment, BXD and E520 cells did not undergo any significant cell death (data not shown).Fig. 3

Bottom Line: Over 60 % of pediatric ependymomas were found to rely on telomerase activity to maintain telomeres, while no ependymomas showed evidence of ALT.Children with telomerase-active tumors had reduced 5-year progression-free survival (29 ± 11 vs 64 ± 18 %; p = 0.03) and overall survival (58 ± 12 vs 83 ± 15 %; p = 0.05) rates compared to those with tumors lacking telomerase activity.Telomerase inhibition represents a promising therapeutic approach for telomerase-active pediatric ependymomas found to characterize high-risk ependymomas.

View Article: PubMed Central - PubMed

Affiliation: The Arthur and Sonia Labatt Brain Tumor Research Centre, The Hospital for Sick Children, Toronto, ON, Canada.

ABSTRACT
Pediatric ependymomas are highly recurrent tumors resistant to conventional chemotherapy. Telomerase, a ribonucleoprotein critical in permitting limitless replication, has been found to be critically important for the maintenance of tumor-initiating cells (TICs). These TICs are chemoresistant, repopulate the tumor from which they are identified, and are drivers of recurrence in numerous cancers. In this study, telomerase enzymatic activity was directly measured and inhibited to assess the therapeutic potential of targeting telomerase. Telomerase repeat amplification protocol (TRAP) (n = 36) and C-circle assay/telomere FISH/ATRX staining (n = 76) were performed on primary ependymomas to determine the prevalence and prognostic potential of telomerase activity or alternative lengthening of telomeres (ALT) as telomere maintenance mechanisms, respectively. Imetelstat, a phase 2 telomerase inhibitor, was used to elucidate the effect of telomerase inhibition on proliferation and tumorigenicity in established cell lines (BXD-1425EPN, R254), a primary TIC line (E520) and xenograft models of pediatric ependymoma. Over 60 % of pediatric ependymomas were found to rely on telomerase activity to maintain telomeres, while no ependymomas showed evidence of ALT. Children with telomerase-active tumors had reduced 5-year progression-free survival (29 ± 11 vs 64 ± 18 %; p = 0.03) and overall survival (58 ± 12 vs 83 ± 15 %; p = 0.05) rates compared to those with tumors lacking telomerase activity. Imetelstat inhibited proliferation and self-renewal by shortening telomeres and inducing senescence in vitro. In vivo, Imetelstat significantly reduced subcutaneous xenograft growth by 40 % (p = 0.03) and completely abolished the tumorigenicity of pediatric ependymoma TICs in an orthotopic xenograft model. Telomerase inhibition represents a promising therapeutic approach for telomerase-active pediatric ependymomas found to characterize high-risk ependymomas.

Show MeSH
Related in: MedlinePlus