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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

The failure of TERT repression in fibroblasts was retained throughout long-term culture.(A), (C) and (D) Relative expression level of TERT, OCT4, COL1A1, and GAPDH in individual hESC clones differentiated to fibroblasts carrying the indicated mutations at days 28, 40, and 45 after differentiation. Expression levels are shown relative to WT hESCs. (B) Average expression of data shown in (A). Expression levels are compared to WT hESC clones. Error bars represent the SEM.DOI:http://dx.doi.org/10.7554/eLife.07918.011
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fig3s2: The failure of TERT repression in fibroblasts was retained throughout long-term culture.(A), (C) and (D) Relative expression level of TERT, OCT4, COL1A1, and GAPDH in individual hESC clones differentiated to fibroblasts carrying the indicated mutations at days 28, 40, and 45 after differentiation. Expression levels are shown relative to WT hESCs. (B) Average expression of data shown in (A). Expression levels are compared to WT hESC clones. Error bars represent the SEM.DOI:http://dx.doi.org/10.7554/eLife.07918.011

Mentions: We differentiated edited hESCs into embryonic bodies (EBs) and eventually into fibroblasts and determined TERT mRNA levels over a 15 day differentiation period (Figure 3A). All cell lines differentiated with equal efficiencies, as evidenced by up-regulation of the differentiation marker COL1A1 and repression of OCT4 transcription (Figure 3B,C). Although TERT expression was successfully down-regulated in cells with wild-type TERT promoter, all three promoter mutation lines retained significant levels of TERT expression (Figure 3A). This failure of TERT transcriptional silencing became apparent as early as 3 days after the induction of differentiation and accumulated into a fourfold increase in TERT expression in cells that carried the −57A/C or the −146C/T mutation and an 8–12-fold increase in cells in which transcription depended on the endogenous TERT promoter with the −124C/T mutation (Figure 3A,B). This failure to appropriately repress TERT transcription during EB differentiation became even more apparent when the cells were differentiated into fibroblast-like cells. As expected, TERT transcription was undetectable in differentiated wild-type fibroblasts. In contrast, fibroblasts with the cancer-associated promoter mutations showed high levels of TERT expression (Figure 3D). This difference was not due to impaired differentiation of cells with the TERT promoter mutations, as these cells had silenced OCT4 and appropriately induced COL1A1 expression (Figure 3D). Importantly, while telomerase activity is not detectable in wild-type fibroblasts, the aberrant TERT expression resulted in robust telomerase activity in fibroblasts that contained the promoter mutations (Figure 3E). As before, we confirmed these findings in an independent TERTΔ/Δ cell line (Figures 3—figure supplement 1A–D) and in individual single cell-derived targeted clones (Figures 3—figure supplement 1E,F). Furthermore we confirmed that this failure to repress TERT expression persists in fibroblasts as late as 45 days after differentiation (Figures 3—figure supplement 2).10.7554/eLife.07918.009Figure 3.Fibroblasts carrying cancer-associated TERT promoter point mutations failed to silence TERT expression upon differentiation and have telomerase activity.


Cancer-associated TERT promoter mutations abrogate telomerase silencing.

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

The failure of TERT repression in fibroblasts was retained throughout long-term culture.(A), (C) and (D) Relative expression level of TERT, OCT4, COL1A1, and GAPDH in individual hESC clones differentiated to fibroblasts carrying the indicated mutations at days 28, 40, and 45 after differentiation. Expression levels are shown relative to WT hESCs. (B) Average expression of data shown in (A). Expression levels are compared to WT hESC clones. Error bars represent the SEM.DOI:http://dx.doi.org/10.7554/eLife.07918.011
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4507476&req=5

fig3s2: The failure of TERT repression in fibroblasts was retained throughout long-term culture.(A), (C) and (D) Relative expression level of TERT, OCT4, COL1A1, and GAPDH in individual hESC clones differentiated to fibroblasts carrying the indicated mutations at days 28, 40, and 45 after differentiation. Expression levels are shown relative to WT hESCs. (B) Average expression of data shown in (A). Expression levels are compared to WT hESC clones. Error bars represent the SEM.DOI:http://dx.doi.org/10.7554/eLife.07918.011
Mentions: We differentiated edited hESCs into embryonic bodies (EBs) and eventually into fibroblasts and determined TERT mRNA levels over a 15 day differentiation period (Figure 3A). All cell lines differentiated with equal efficiencies, as evidenced by up-regulation of the differentiation marker COL1A1 and repression of OCT4 transcription (Figure 3B,C). Although TERT expression was successfully down-regulated in cells with wild-type TERT promoter, all three promoter mutation lines retained significant levels of TERT expression (Figure 3A). This failure of TERT transcriptional silencing became apparent as early as 3 days after the induction of differentiation and accumulated into a fourfold increase in TERT expression in cells that carried the −57A/C or the −146C/T mutation and an 8–12-fold increase in cells in which transcription depended on the endogenous TERT promoter with the −124C/T mutation (Figure 3A,B). This failure to appropriately repress TERT transcription during EB differentiation became even more apparent when the cells were differentiated into fibroblast-like cells. As expected, TERT transcription was undetectable in differentiated wild-type fibroblasts. In contrast, fibroblasts with the cancer-associated promoter mutations showed high levels of TERT expression (Figure 3D). This difference was not due to impaired differentiation of cells with the TERT promoter mutations, as these cells had silenced OCT4 and appropriately induced COL1A1 expression (Figure 3D). Importantly, while telomerase activity is not detectable in wild-type fibroblasts, the aberrant TERT expression resulted in robust telomerase activity in fibroblasts that contained the promoter mutations (Figure 3E). As before, we confirmed these findings in an independent TERTΔ/Δ cell line (Figures 3—figure supplement 1A–D) and in individual single cell-derived targeted clones (Figures 3—figure supplement 1E,F). Furthermore we confirmed that this failure to repress TERT expression persists in fibroblasts as late as 45 days after differentiation (Figures 3—figure supplement 2).10.7554/eLife.07918.009Figure 3.Fibroblasts carrying cancer-associated TERT promoter point mutations failed to silence TERT expression upon differentiation and have 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