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Cancer-associated TERT promoter mutations abrogate telomerase silencing.

Chiba K, Johnson JZ, Vogan JM, Wagner T, Boyle JM, Hockemeyer D - Elife (2015)

Bottom Line: We used genome editing of human pluripotent stem cells with physiological telomerase expression to elucidate the mechanism by which these mutations contribute to human disease.Surprisingly, telomerase-expressing embryonic stem cells engineered to carry any of the three most frequent TERT promoter mutations showed only a modest increase in TERT transcription with no impact on telomerase activity.Thus, TERT promoter mutations are sufficient to overcome the proliferative barrier imposed by telomere shortening without additional tumor-selected mutations.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.

ABSTRACT
Mutations in the human telomerase reverse transcriptase (TERT) promoter are the most frequent non-coding mutations in cancer, but their molecular mechanism in tumorigenesis has not been established. We used genome editing of human pluripotent stem cells with physiological telomerase expression to elucidate the mechanism by which these mutations contribute to human disease. Surprisingly, telomerase-expressing embryonic stem cells engineered to carry any of the three most frequent TERT promoter mutations showed only a modest increase in TERT transcription with no impact on telomerase activity. However, upon differentiation into somatic cells, which normally silence telomerase, cells with TERT promoter mutations failed to silence TERT expression, resulting in increased telomerase activity and aberrantly long telomeres. Thus, TERT promoter mutations are sufficient to overcome the proliferative barrier imposed by telomere shortening without additional tumor-selected mutations. These data establish that TERT promoter mutations can promote immortalization and tumorigenesis of incipient cancer cells.

No MeSH data available.


Related in: MedlinePlus

Clonal analysis of TERT promoter mutation NPCs confirmed results from bulk analysis.(A) Quantitative RT-PCR of TERT, OCT4, NESTIN, and GAPDH in NPCs differentiated from individual clones of the targeted hESCs (28 days after differentiation from hESCs). Expression levels are shown relative to WT hESCs. (B) Average expression of data shown in (A). Expression levels are compared to WT hESCs. Error bars represent the SEM.DOI:http://dx.doi.org/10.7554/eLife.07918.013
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fig4s1: Clonal analysis of TERT promoter mutation NPCs confirmed results from bulk analysis.(A) Quantitative RT-PCR of TERT, OCT4, NESTIN, and GAPDH in NPCs differentiated from individual clones of the targeted hESCs (28 days after differentiation from hESCs). Expression levels are shown relative to WT hESCs. (B) Average expression of data shown in (A). Expression levels are compared to WT hESCs. Error bars represent the SEM.DOI:http://dx.doi.org/10.7554/eLife.07918.013

Mentions: The failure to silence telomerase could be specific to the fibroblast differentiation paradigm or a more general defect during differentiation. To address this issue, we first generated NPCs using the highly robust dual SMAD inhibition protocol (Chambers et al., 2009), establishing NPCs that can be maintained in culture for extended periods of time with low levels of telomerase expression. These NPCs can be further differentiated towards terminally differentiated non-proliferating post mitotic neurons that are characterized by the expression of the pan-neural marker proteins TUJ1 and NEUN. Independent of their genotype, all cells were able to differentiate into NPCs and neurons showing equal down-regulation of OCT4 expression and induction of neuronal marker genes (Figure 4A–C). However, a striking difference became apparent in TERT levels as both NPCs and neurons that carried the TERT promoter mutations failed to repress TERT transcription (Figure 4A,C and Figures 4—figure supplement 1) and showed robust telomerase activity (Figure 4D). Even when neurons were maintained in the presence of a mitotic inhibitor, the promoter mutations led to elevated TERT mRNA and telomerase activity levels, suggesting that telomerase activity can accumulate in slowly and non-dividing cells as late as 1 month after induction of terminal differentiation (Figure 4C,D).10.7554/eLife.07918.012Figure 4.Neural precursors and neurons differentiated from the promoter mutation hESCs failed to repress TERT and telomerase activity.


Cancer-associated TERT promoter mutations abrogate telomerase silencing.

Chiba K, Johnson JZ, Vogan JM, Wagner T, Boyle JM, Hockemeyer D - Elife (2015)

Clonal analysis of TERT promoter mutation NPCs confirmed results from bulk analysis.(A) Quantitative RT-PCR of TERT, OCT4, NESTIN, and GAPDH in NPCs differentiated from individual clones of the targeted hESCs (28 days after differentiation from hESCs). Expression levels are shown relative to WT hESCs. (B) Average expression of data shown in (A). Expression levels are compared to WT hESCs. Error bars represent the SEM.DOI:http://dx.doi.org/10.7554/eLife.07918.013
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4507476&req=5

fig4s1: Clonal analysis of TERT promoter mutation NPCs confirmed results from bulk analysis.(A) Quantitative RT-PCR of TERT, OCT4, NESTIN, and GAPDH in NPCs differentiated from individual clones of the targeted hESCs (28 days after differentiation from hESCs). Expression levels are shown relative to WT hESCs. (B) Average expression of data shown in (A). Expression levels are compared to WT hESCs. Error bars represent the SEM.DOI:http://dx.doi.org/10.7554/eLife.07918.013
Mentions: The failure to silence telomerase could be specific to the fibroblast differentiation paradigm or a more general defect during differentiation. To address this issue, we first generated NPCs using the highly robust dual SMAD inhibition protocol (Chambers et al., 2009), establishing NPCs that can be maintained in culture for extended periods of time with low levels of telomerase expression. These NPCs can be further differentiated towards terminally differentiated non-proliferating post mitotic neurons that are characterized by the expression of the pan-neural marker proteins TUJ1 and NEUN. Independent of their genotype, all cells were able to differentiate into NPCs and neurons showing equal down-regulation of OCT4 expression and induction of neuronal marker genes (Figure 4A–C). However, a striking difference became apparent in TERT levels as both NPCs and neurons that carried the TERT promoter mutations failed to repress TERT transcription (Figure 4A,C and Figures 4—figure supplement 1) and showed robust telomerase activity (Figure 4D). Even when neurons were maintained in the presence of a mitotic inhibitor, the promoter mutations led to elevated TERT mRNA and telomerase activity levels, suggesting that telomerase activity can accumulate in slowly and non-dividing cells as late as 1 month after induction of terminal differentiation (Figure 4C,D).10.7554/eLife.07918.012Figure 4.Neural precursors and neurons differentiated from the promoter mutation hESCs failed to repress TERT and telomerase activity.

Bottom Line: We used genome editing of human pluripotent stem cells with physiological telomerase expression to elucidate the mechanism by which these mutations contribute to human disease.Surprisingly, telomerase-expressing embryonic stem cells engineered to carry any of the three most frequent TERT promoter mutations showed only a modest increase in TERT transcription with no impact on telomerase activity.Thus, TERT promoter mutations are sufficient to overcome the proliferative barrier imposed by telomere shortening without additional tumor-selected mutations.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.

ABSTRACT
Mutations in the human telomerase reverse transcriptase (TERT) promoter are the most frequent non-coding mutations in cancer, but their molecular mechanism in tumorigenesis has not been established. We used genome editing of human pluripotent stem cells with physiological telomerase expression to elucidate the mechanism by which these mutations contribute to human disease. Surprisingly, telomerase-expressing embryonic stem cells engineered to carry any of the three most frequent TERT promoter mutations showed only a modest increase in TERT transcription with no impact on telomerase activity. However, upon differentiation into somatic cells, which normally silence telomerase, cells with TERT promoter mutations failed to silence TERT expression, resulting in increased telomerase activity and aberrantly long telomeres. Thus, TERT promoter mutations are sufficient to overcome the proliferative barrier imposed by telomere shortening without additional tumor-selected mutations. These data establish that TERT promoter mutations can promote immortalization and tumorigenesis of incipient cancer cells.

No MeSH data available.


Related in: MedlinePlus