Common Telomere Changes during In Vivo Reprogramming and Early Stages of Tumorigenesis

Abstract

Reprogramming of differentiated cells into induced pluripotent stem cells has been recently achieved in vivo in mice. Telomeres are essential for chromosomal stability and determine organismal life span as well as cancer growth. Here, we study whether tissue dedifferentiation induced by in vivo reprogramming involves changes at telomeres. We find telomerase-dependent telomere elongation in the reprogrammed areas. Notably, we found highly upregulated expression of the TRF1 telomere protein in the reprogrammed areas, which was independent of telomere length. Moreover, TRF1 inhibition reduced in vivo reprogramming efficiency. Importantly, we extend the finding of TRF1 upregulation to pathological tissue dedifferentiation associated with neoplasias, in particular during pancreatic acinar-to-ductal metaplasia, a process that involves transdifferentiation of adult acinar cells into ductal-like cells due to K-Ras oncogene expression. These findings place telomeres as important players in cellular plasticity both during in vivo reprogramming and in pathological conditions associated with increased plasticity, such as cancer.

Keywords: ADM; TRF1; cancer; cellular plasticity; in vivo reprogramming; regeneration; stem cells; telomeres; transdifferentiation; tumorigenesis.

Figure 1

Telomeres Elongate during In Vivo Reprogramming (A) Schematic representation of the induction of in vivo reprogramming in i4F mice. (B and C) Left: representative immuno-FISH images showing OCT4 (green) and telomeres (red) in (B) the large intestine and (C) the pancreas of reprogrammable mice after induction of in vivo reprogramming. White dotted lines mark the reprogrammed areas. Scale bars, 25 μm. Right: quantification of telomere signal in in vivo reprogrammed cells and the corresponding non-reprogrammed control cells. Error bars denote SE. Statistical analysis by Student's t test. n, number of nuclei. Number of mice analyzed = 3. See also Figures S1, S4, and S5.

Figure 2

TERC Expression Increases during In Vivo Reprogramming (A) Representative images corresponding to TERC detection by RNA-FISH (red) followed by immunostaining to detect OCT4 (green) in the pancreas of induced reprogrammable mice of the indicated genotypes. Arrowheads indicate the presence of TERC foci. Scale bars, 25 μm. Zoomed areas (orange boxes) of TERC detection are shown on the right. Scale bars, 50 μm. White dashed lines contain OCT4-positive regions. (B) Number of TERC spots per nuclei. Expression of TERC is increased in the reprogrammed areas. Error bars denote SE. Statistical analysis by Student's t test. Number of mice analyzed is indicated. (C) Expression of TERC, TRF1, and OCT4 in non-reprogrammed pancreas compared with in vivo iPSCs of pancreatic origin as measured by qRT-PCR. Expression of these genes is strongly upregulated during the acquisition of pluripotency in vivo. Error bars denote SE. Statistical analysis by Student's t test. n, number of independent pancreas or in vivo iPSC clones obtained from independent reprogrammed pancreas, respectively. See also Figures S1 and S2.

Figure 3

Telomere Elongation during In Vivo Reprogramming Depends on Telomerase Activity (A and B) Left: representative immuno-FISH images showing OCT4 (green) and telomeres (red) in (A) the stomach and (B) the pancreas of i4F G1Terc^−/− reprogrammable mice after induction of in vivo reprogramming. White dashed lines mark the reprogrammed areas. Scale bars, 25 μm. Right: quantification of telomere signal in in vivo reprogrammed cells and the corresponding non-reprogrammed control cells. Telomere lengthening during in vivo reprogramming is abolished in the absence of telomerase activity. Error bars denote SE. n, number of nuclei. Number of mice analyzed = 3. (C) Comparison of telomere lengthening during in vivo reprogramming in i4F and i4F G1Terc^−/− reprogrammable mice. Note that most of the telomere elongation observed during in vivo reprogramming is dependent on telomerase activity. Error bars denote SE. Statistical analysis by Student's t test. n, number of tissues from independent mice analyzed. See also Figure S1.

Figure 4

Increased TRF1 Expression during In Vivo Reprogramming Correlates with OCT4 Expression and Is Uncoupled from Telomere Length (A) Upper: representative images of double immunofluorescence against OCT4 (red) and TRF1 (green) proteins in (left) the large intestine and (right) the pancreas of reprogrammable mice after induction of in vivo reprogramming. A strong correlation between the presence of OCT4 and high TRF1 expression can be observed. Lower: quantification of TRF1 expression in in vivo reprogrammed cells and the corresponding non-reprogrammed control cells. Scale bars, 25 μm. Error bars denote SE. Statistical analysis by Student's t test. n, number of nuclei. Number of mice analyzed = 3. (B) Upper: representative images of double immunofluorescence against OCT4 (red) and TRF1 (green) proteins in (left) the stomach and (right) the pancreas of i4F G1Terc^−/− reprogrammable mice after induction of in vivo reprogramming. High TRF1 levels are observed in the in vivo reprogrammed areas even in the absence of a functional telomerase enzyme. Lower: quantification of TRF1 expression in in vivo reprogrammed cells and the corresponding non-reprogrammed control cells. White dashed lines mark the reprogrammed area. Scale bars, 25 μm. Error bars denote SE. Statistical analysis by Student's t test. n, number of nuclei. Number of mice analyzed = 3. See also Figures S1 and S3.

Figure 5

In Vivo Treatment with TRF1 Inhibitor ETP-47037 Reduces Dysplasia during In Vivo Reprogramming without Affecting Telomere Length (A) Representative images of pancreas, stomach, and large intestine from eGFP-TRF1^+/KIi4F mice in vivo treated with vehicle (top) or ETP-47037 (bottom) compound during induction of in vivo reprogramming for 7 days with high doxycycline. Arrowheads point to foci of dysplasia. Treatment with TRF1 inhibitor reduces the dysplasia associated with in vivo reprogramming. Scale bars, 100 μm. (B) Quantification of the percentage of dysplastic area in the large intestine of i4F mice treated with vehicle or TRF1 inhibitor ETP-47037. Note the strong reduction in the affected area in mice treated with ETP-47037. Error bars denote SE. Statistical analysis by Student's t test. n, number of mice analyzed in each group. (C) Quantification of telomere length in normal non-dysplastic cells of pancreas from induced eGFP-TRF1^+/KIi4F mice and i4F mice. Inhibition of TRF1 does not alter telomere length in normal tissue. Error bars denote SE. Statistical analysis by Student's t test. n, number of nuclei. Number of mice analyzed in each group = 2. n.s., not significant. (D) Quantification of telomere elongation of dysplastic cells, compared with non-dysplastic cells from the same tissue, in mice treated with vehicle or TRF1 inhibitor ETP-47037. Inhibition of TRF1 does not change the telomere length increase of the dysplastic cells. Error bars denote SE. Statistical analysis by Student's t test. n, number of mice analyzed in each group. n.s., not significant.

Figure 6

Increased TRF1 Expression in Acinar-to-Ductal Metaplasias and PanINs of ElasK-Ras^G12V Mice (A) Left: representative images of pancreatic lesions (metaplasias and PanINs) of ElasK-Ras^G12V mice. Serial sections of pancreas from ElasK-Ras^G12V mice were stained with H&E and immunostained with cytokeratin 19 (CK19). Right: zoomed panel of the selected areas showing double immunofluorescence against CK19 (red) and TRF1 (green). White dashed lines mark pancreatic lesions. Orange dashed lines mark normal acinar cells used as control. Elevated TRF1 protein expression when compared with normal acinar cells can be detected in all the metaplasias analyzed (white arrowhead) and in most of the PanINs (pink arrowhead). A fraction of the PanINs analyzed did not show increased TRF1 levels (yellow arrowheads). Scale bars, 25 μm. (B) Quantification of TRF1 expression in acinar-to-ductal metaplasias and PanINs and their corresponding normal acinar control cells. TRF1 levels are significantly elevated in metaplasias (left) and in a fraction of the PanINs analyzed (right). Error bars denote SE. Statistical analysis by Student's t test. n, number of pancreatic lesions analyzed. Number of mice analyzed = 4. (C) Double immunofluorescence against CK19 and OCT4 in a pancreatic lesion expressing elevated levels of TRF1 (see A). OCT4 expression is not detected in the dedifferentiated acinar cells. Scale bars, 25 μm. See also Figure S6.

Figure 7

Model for the Role of In Vivo Reprogramming in Tissue Repair and Cancer Initiation Adult stem cells within a tissue show the longest telomeres, telomerase activity, and high levels of TRF1 protein. As they mobilize for tissue renewal, telomeres shorten, telomerase activity is lost, and TRF1 expression diminishes. In vivo reprogramming reverses these features generating dedifferentiated cells with longer telomeres, telomerase expression, and higher levels of TRF1. The similarities of the in vivo reprogrammed cells to the adult stem cells suggest the possibility that reprogrammed cells could have a role in tissue regeneration. Other cellular plastic processes, such as dedifferentiation associated with initiation of tumorigenesis, also show increased levels of TRF1 protein and elongation of telomeres, indicating a general role of these telomeric events in processes associated with cell fate change.