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Crucial function of histone deacetylase 1 for differentiation of teratomas in mice and humans.

Lagger S, Meunier D, Mikula M, Brunmeir R, Schlederer M, Artaker M, Pusch O, Egger G, Hagelkruys A, Mikulits W, Weitzer G, Muellner EW, Susani M, Kenner L, Seiser C - EMBO J. (2010)

Bottom Line: However, the specific HDAC isoforms that mediate these effects are not yet identified.Surprisingly, loss of HDAC1 was not only linked to increased apoptosis, but also to significantly enhanced proliferation.These findings reveal a novel role for HDAC1 in the control of tumour proliferation and identify HDAC1 as potential marker for benign teratomas.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical University of Vienna, Vienna Biocenter, Vienna, Austria.

ABSTRACT
Histone deacetylase (HDAC) inhibitors induce cell cycle arrest, differentiation or apoptosis in tumour cells and are, therefore, promising anti-cancer reagents. However, the specific HDAC isoforms that mediate these effects are not yet identified. To explore the role of HDAC1 in tumourigenesis and tumour proliferation, we established an experimental teratoma model using wild-type and HDAC1-deficient embryonic stem cells. HDAC1-deficient teratomas showed no significant difference in size compared with wild-type teratomas. Surprisingly, loss of HDAC1 was not only linked to increased apoptosis, but also to significantly enhanced proliferation. Epithelial structures showed reduced differentiation as monitored by Oct3/4 expression and changed E-cadherin localization and displayed up-regulated expression of SNAIL1, a regulator of epithelial cell plasticity. Increased levels of the transcriptional regulator SNAIL1 are crucial for enhanced proliferation and reduced differentiation of HDAC1-deficient teratoma. Importantly, the analysis of human teratomas revealed a similar link between loss of HDAC1 and enhanced tumour malignancy. These findings reveal a novel role for HDAC1 in the control of tumour proliferation and identify HDAC1 as potential marker for benign teratomas.

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SNAIL1 as crucial mediator of the phenotype of HDAC1−/− teratomas. (A) shRNA-mediated silencing of SNAIL1 in HDAC1+/+ and HDAC1−/− ES cells. Expression of SNAIL1 protein upon stable expression of a non-targeting control shRNA (NT) or two different SNAIL1 shRNAs (SN1-1 and SN1-2) in HDAC1+/+ and HDAC1−/− ES cells was analysed on western blots. The membranes were probed with antibodies against SNAIL1 and Actin as loading control. (B–E) ES cells shown in (A) were subcutaneously injected in SCID/Balb/c mice and teratoma formation as well as tumour size was monitored every 4 days. Recipient SCID mice were killed after 20 days post-injection, and teratomas were removed and analysed. (B) Statistical comparison of the tumour volume of NT controls (black bars) and SNAIL1 knockdown (SN1) teratomas (white bars). The tumour volume (mm3) was calculated using the formula ‘(width2 × length) × ½'. (C) Quantification of IHC analysis of representative HDAC1+/+ and HDAC1−/− control (NT) and SNAIL1 (SN1) knockdown teratoma paraffin sections with SNAIL1 antibody. The intensities of SNAIL1 positively stained cells were evaluated by the HistoQuest Software and are shown separately for HDAC1+/+ and HDAC1−/− tumours. (D, E) IHC analysis of representative HDAC1−/− control (HDAC1−/−NT) and SNAIL1 knockdown teratoma (HDAC1−/−SN1) paraffin sections with antibodies specific for E-cadherin (D) and Ki67 (E). The nuclei were counterstained with Mayer's hemalaun (blue staining). All pictures were taken in a × 20 magnification. (D) Patches with cytosolic E-cadherin staining were evaluated by the HistoQuest Software as shown in the graphs on the right. (E) Quantification of Ki67-positive cells is shown in the graph on the right. *P<0.05; **P<0.01; ***P<0.001.
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f5: SNAIL1 as crucial mediator of the phenotype of HDAC1−/− teratomas. (A) shRNA-mediated silencing of SNAIL1 in HDAC1+/+ and HDAC1−/− ES cells. Expression of SNAIL1 protein upon stable expression of a non-targeting control shRNA (NT) or two different SNAIL1 shRNAs (SN1-1 and SN1-2) in HDAC1+/+ and HDAC1−/− ES cells was analysed on western blots. The membranes were probed with antibodies against SNAIL1 and Actin as loading control. (B–E) ES cells shown in (A) were subcutaneously injected in SCID/Balb/c mice and teratoma formation as well as tumour size was monitored every 4 days. Recipient SCID mice were killed after 20 days post-injection, and teratomas were removed and analysed. (B) Statistical comparison of the tumour volume of NT controls (black bars) and SNAIL1 knockdown (SN1) teratomas (white bars). The tumour volume (mm3) was calculated using the formula ‘(width2 × length) × ½'. (C) Quantification of IHC analysis of representative HDAC1+/+ and HDAC1−/− control (NT) and SNAIL1 (SN1) knockdown teratoma paraffin sections with SNAIL1 antibody. The intensities of SNAIL1 positively stained cells were evaluated by the HistoQuest Software and are shown separately for HDAC1+/+ and HDAC1−/− tumours. (D, E) IHC analysis of representative HDAC1−/− control (HDAC1−/−NT) and SNAIL1 knockdown teratoma (HDAC1−/−SN1) paraffin sections with antibodies specific for E-cadherin (D) and Ki67 (E). The nuclei were counterstained with Mayer's hemalaun (blue staining). All pictures were taken in a × 20 magnification. (D) Patches with cytosolic E-cadherin staining were evaluated by the HistoQuest Software as shown in the graphs on the right. (E) Quantification of Ki67-positive cells is shown in the graph on the right. *P<0.05; **P<0.01; ***P<0.001.

Mentions: Given the aforementioned role of SNAIL1 in tumourigenesis, we next examined whether SNAIL1 contributes to the phenotype of HDAC1-deficient teratomas. We infected HDAC1+/+ and HDAC1−/− ES cells with two lentiviral vectors containing two different shRNAs targeting Snail1 and corresponding non-target controls. In contrast to teratomas and embryonal carcinoma cells, SNAIL1 expression is not enhanced in HDAC1−/− mouse ES cells (Figure 5A). Expression of SNAIL1 shRNAs resulted in significant reduction in SNAIL1 protein levels, whereas mismatch and non-target control shRNAs had no effect. Two different SNAIL1 knockdown cell lines and corresponding control cells were subcutaneously injected in SCID/BALBc female mice to create teratomas. Control teratomas (HDAC1+/+ NT and HDAC1−/− NT) were comparable with the HDAC1+/+ and HDAC−/− tumours described above. As previously observed for epidermal carcinomas (Olmeda et al, 2007), silencing of SNAIL1 had a strong effect on teratoma formation. SNAIL1 knockdown resulted in a 61% reduction for HDAC1+/+ and 81% reduction for HDAC1−/− teratomas in tumour volume compared with the control teratomas (NT) 20 days after injection (Figure 5B). Quantification of SNAIL1-positive cells and their signal intensity revealed a strong reduction in SNAIL1 expression upon SNAIL1 knockdown in both HDAC1+/+ and HDAC1−/− teratomas (Figure 5C). Importantly, knockdown of SNAIL1 resulted in loss of the hallmarks of HDAC1-deficient teratomas, namely delocalised cytosolic E-cadherin and increased cell proliferation. As shown in Figures 5D and E, SNAIL1 knockdown in HDAC1−/− teratomas significantly reduced the presence of patches with cytosolic E-cadherin staining and the number of highly proliferating Ki67-positive cells. These results suggest that SNAIL1 strongly contributes to the phenotype of HDAC1−/− teratomas.


Crucial function of histone deacetylase 1 for differentiation of teratomas in mice and humans.

Lagger S, Meunier D, Mikula M, Brunmeir R, Schlederer M, Artaker M, Pusch O, Egger G, Hagelkruys A, Mikulits W, Weitzer G, Muellner EW, Susani M, Kenner L, Seiser C - EMBO J. (2010)

SNAIL1 as crucial mediator of the phenotype of HDAC1−/− teratomas. (A) shRNA-mediated silencing of SNAIL1 in HDAC1+/+ and HDAC1−/− ES cells. Expression of SNAIL1 protein upon stable expression of a non-targeting control shRNA (NT) or two different SNAIL1 shRNAs (SN1-1 and SN1-2) in HDAC1+/+ and HDAC1−/− ES cells was analysed on western blots. The membranes were probed with antibodies against SNAIL1 and Actin as loading control. (B–E) ES cells shown in (A) were subcutaneously injected in SCID/Balb/c mice and teratoma formation as well as tumour size was monitored every 4 days. Recipient SCID mice were killed after 20 days post-injection, and teratomas were removed and analysed. (B) Statistical comparison of the tumour volume of NT controls (black bars) and SNAIL1 knockdown (SN1) teratomas (white bars). The tumour volume (mm3) was calculated using the formula ‘(width2 × length) × ½'. (C) Quantification of IHC analysis of representative HDAC1+/+ and HDAC1−/− control (NT) and SNAIL1 (SN1) knockdown teratoma paraffin sections with SNAIL1 antibody. The intensities of SNAIL1 positively stained cells were evaluated by the HistoQuest Software and are shown separately for HDAC1+/+ and HDAC1−/− tumours. (D, E) IHC analysis of representative HDAC1−/− control (HDAC1−/−NT) and SNAIL1 knockdown teratoma (HDAC1−/−SN1) paraffin sections with antibodies specific for E-cadherin (D) and Ki67 (E). The nuclei were counterstained with Mayer's hemalaun (blue staining). All pictures were taken in a × 20 magnification. (D) Patches with cytosolic E-cadherin staining were evaluated by the HistoQuest Software as shown in the graphs on the right. (E) Quantification of Ki67-positive cells is shown in the graph on the right. *P<0.05; **P<0.01; ***P<0.001.
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f5: SNAIL1 as crucial mediator of the phenotype of HDAC1−/− teratomas. (A) shRNA-mediated silencing of SNAIL1 in HDAC1+/+ and HDAC1−/− ES cells. Expression of SNAIL1 protein upon stable expression of a non-targeting control shRNA (NT) or two different SNAIL1 shRNAs (SN1-1 and SN1-2) in HDAC1+/+ and HDAC1−/− ES cells was analysed on western blots. The membranes were probed with antibodies against SNAIL1 and Actin as loading control. (B–E) ES cells shown in (A) were subcutaneously injected in SCID/Balb/c mice and teratoma formation as well as tumour size was monitored every 4 days. Recipient SCID mice were killed after 20 days post-injection, and teratomas were removed and analysed. (B) Statistical comparison of the tumour volume of NT controls (black bars) and SNAIL1 knockdown (SN1) teratomas (white bars). The tumour volume (mm3) was calculated using the formula ‘(width2 × length) × ½'. (C) Quantification of IHC analysis of representative HDAC1+/+ and HDAC1−/− control (NT) and SNAIL1 (SN1) knockdown teratoma paraffin sections with SNAIL1 antibody. The intensities of SNAIL1 positively stained cells were evaluated by the HistoQuest Software and are shown separately for HDAC1+/+ and HDAC1−/− tumours. (D, E) IHC analysis of representative HDAC1−/− control (HDAC1−/−NT) and SNAIL1 knockdown teratoma (HDAC1−/−SN1) paraffin sections with antibodies specific for E-cadherin (D) and Ki67 (E). The nuclei were counterstained with Mayer's hemalaun (blue staining). All pictures were taken in a × 20 magnification. (D) Patches with cytosolic E-cadherin staining were evaluated by the HistoQuest Software as shown in the graphs on the right. (E) Quantification of Ki67-positive cells is shown in the graph on the right. *P<0.05; **P<0.01; ***P<0.001.
Mentions: Given the aforementioned role of SNAIL1 in tumourigenesis, we next examined whether SNAIL1 contributes to the phenotype of HDAC1-deficient teratomas. We infected HDAC1+/+ and HDAC1−/− ES cells with two lentiviral vectors containing two different shRNAs targeting Snail1 and corresponding non-target controls. In contrast to teratomas and embryonal carcinoma cells, SNAIL1 expression is not enhanced in HDAC1−/− mouse ES cells (Figure 5A). Expression of SNAIL1 shRNAs resulted in significant reduction in SNAIL1 protein levels, whereas mismatch and non-target control shRNAs had no effect. Two different SNAIL1 knockdown cell lines and corresponding control cells were subcutaneously injected in SCID/BALBc female mice to create teratomas. Control teratomas (HDAC1+/+ NT and HDAC1−/− NT) were comparable with the HDAC1+/+ and HDAC−/− tumours described above. As previously observed for epidermal carcinomas (Olmeda et al, 2007), silencing of SNAIL1 had a strong effect on teratoma formation. SNAIL1 knockdown resulted in a 61% reduction for HDAC1+/+ and 81% reduction for HDAC1−/− teratomas in tumour volume compared with the control teratomas (NT) 20 days after injection (Figure 5B). Quantification of SNAIL1-positive cells and their signal intensity revealed a strong reduction in SNAIL1 expression upon SNAIL1 knockdown in both HDAC1+/+ and HDAC1−/− teratomas (Figure 5C). Importantly, knockdown of SNAIL1 resulted in loss of the hallmarks of HDAC1-deficient teratomas, namely delocalised cytosolic E-cadherin and increased cell proliferation. As shown in Figures 5D and E, SNAIL1 knockdown in HDAC1−/− teratomas significantly reduced the presence of patches with cytosolic E-cadherin staining and the number of highly proliferating Ki67-positive cells. These results suggest that SNAIL1 strongly contributes to the phenotype of HDAC1−/− teratomas.

Bottom Line: However, the specific HDAC isoforms that mediate these effects are not yet identified.Surprisingly, loss of HDAC1 was not only linked to increased apoptosis, but also to significantly enhanced proliferation.These findings reveal a novel role for HDAC1 in the control of tumour proliferation and identify HDAC1 as potential marker for benign teratomas.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical University of Vienna, Vienna Biocenter, Vienna, Austria.

ABSTRACT
Histone deacetylase (HDAC) inhibitors induce cell cycle arrest, differentiation or apoptosis in tumour cells and are, therefore, promising anti-cancer reagents. However, the specific HDAC isoforms that mediate these effects are not yet identified. To explore the role of HDAC1 in tumourigenesis and tumour proliferation, we established an experimental teratoma model using wild-type and HDAC1-deficient embryonic stem cells. HDAC1-deficient teratomas showed no significant difference in size compared with wild-type teratomas. Surprisingly, loss of HDAC1 was not only linked to increased apoptosis, but also to significantly enhanced proliferation. Epithelial structures showed reduced differentiation as monitored by Oct3/4 expression and changed E-cadherin localization and displayed up-regulated expression of SNAIL1, a regulator of epithelial cell plasticity. Increased levels of the transcriptional regulator SNAIL1 are crucial for enhanced proliferation and reduced differentiation of HDAC1-deficient teratoma. Importantly, the analysis of human teratomas revealed a similar link between loss of HDAC1 and enhanced tumour malignancy. These findings reveal a novel role for HDAC1 in the control of tumour proliferation and identify HDAC1 as potential marker for benign teratomas.

Show MeSH
Related in: MedlinePlus