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Inhibition of Six1 affects tumour invasion and the expression of cancer stem cell markers in pancreatic cancer

View Article: PubMed Central - PubMed

ABSTRACT

Background: Epithelial-to-mesenchymal transition (EMT) and cancer stem cells (CSC) contribute to tumour progression and metastasis. Assessment of transcription factors involved in these two mechanisms can help to identify new targets for an oncological therapy. In this study, we focused on the evaluation of the transcription factor Six1 (Sine oculis 1). This protein is involved in embryologic development and its contribution to carcinogenesis has been described in several studies.

Methods: Immunohistochemistry against Six1 was performed on a tissue microarray containing specimens of primary pancreatic ductal adenocarcinomas (PDAC) of 139 patients. Nuclear and cytoplasmic expression was evaluated and correlated to histopathological parameters. Expression of Six1 was inhibited transiently by siRNA in Panc1 and BxPc3 cells and stably by shRNA in Panc1 cells. Expression analysis of CDH1 and Vimentin mRNA was performed and cell motility was tested in a migration assay. Panc1 cells transfected with Six1 shRNA or scrambled shRNA were injected subcutaneously into nude mice. Tumour growth was observed for four weeks. Afterwards, tumours were stained against Six1, CD24 and CD44.

Results: Six1 was overexpressed in the cytoplasm and cellular nuclei in malignant tissues (p < 0.0001). No correlation to histopathological parameters could be detected. Six1 down-regulation decreased pancreatic cancer cell motility in vitro. CDH1 and vimentin expression was decreased after inhibition of the expression of Six1. Pancreatic tumours with impaired expression of Six1 showed significantly delayed growth and displayed loss of the CD24+/CD44+ phenotype.

Conclusion: We show that Six1 is overexpressed in human PDAC and that its inhibition results in a decreased tumour progression in vitro and in vivo. Therefore, targeting Six1 might be a novel therapeutic approach in patients with pancreatic cancer.

Electronic supplementary material: The online version of this article (doi:10.1186/s12885-017-3225-5) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

Six1 downregulation results in a growth arrest of Panc1 cells in a xenograft model. a Body weight curve of mice. Straight line: Panc-1 tumours with scramble shRNA (Panc1shCtl). Dashed line: Panc-1 tumours with Six1-shRNA (Panc1shSix1). No difference in body weight in both groups. b Tumour growth curve of Panc-1 tumours. Straight line: Panc-1 tumours with scramble shRNA (Panc1shCtl). Dashed line: Panc-1 tumours with Six1-shRNA (Panc1shSix1). c Tumour volume of Panc-1 tumour after resection from xenograft models. Upper panel: Panc-1 tumours with Six1-shRNA (Panc1shSix1). Lower panel: Panc-1 tumours with scramble shRNA (Panc1shCtl). d Representative figures for expression of Six1 in Panc-1 tumours with scramble shRNA (left panel) and tumours with Six1-shRNA (right panel). e,f Representative figures for expression of CD44 e and CD24 f in Panc-1 tumours with scramble shRNA (left panel) and tumours with Six1-shRNA (right panel)
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Fig3: Six1 downregulation results in a growth arrest of Panc1 cells in a xenograft model. a Body weight curve of mice. Straight line: Panc-1 tumours with scramble shRNA (Panc1shCtl). Dashed line: Panc-1 tumours with Six1-shRNA (Panc1shSix1). No difference in body weight in both groups. b Tumour growth curve of Panc-1 tumours. Straight line: Panc-1 tumours with scramble shRNA (Panc1shCtl). Dashed line: Panc-1 tumours with Six1-shRNA (Panc1shSix1). c Tumour volume of Panc-1 tumour after resection from xenograft models. Upper panel: Panc-1 tumours with Six1-shRNA (Panc1shSix1). Lower panel: Panc-1 tumours with scramble shRNA (Panc1shCtl). d Representative figures for expression of Six1 in Panc-1 tumours with scramble shRNA (left panel) and tumours with Six1-shRNA (right panel). e,f Representative figures for expression of CD44 e and CD24 f in Panc-1 tumours with scramble shRNA (left panel) and tumours with Six1-shRNA (right panel)

Mentions: We assessed the effect of Six1 inhibition in Panc1 cells in vivo in a xenograft model. For this analysis, shRNA-transfected Panc1 cells were used, showing a stably decreased expression of Six1. After injection of tumour cells into nude mice (2.5 × 106 cells/mouse), body weight and tumour growth were observed for four weeks (Fig. 3a–c). The body weight did not show any difference between these two groups during the observation time. In the beginning, the average tumour volume was 47.65 mm3 (±19.34) in the control group (Panc1shctrl) and 47.00 mm3 (±15.90) in the group with Six1-shRNA (Panc1shSix1). After two weeks, the tumour volume had increased to 86.78 mm3 (±33.93) in the control group and had declined to 39.84 mm3 (±18.54) in Panc1shSix1group (p = 0.0018). At time of euthanasia, the average tumour volume was 124.13 mm3 (±46.59) in the Panc1shctrl group and 50.22 mm3 (±29.76) in Panc1shSix1 (p = 0.0008). After euthanasia, tumour samples of both groups were immunostained against Six1. As expected, tumours from the Panc1shctrl group showed a higher expression of Six1 than tumours from the Panc1shSix1 group (Fig. 3d). Interestingly, in the tumour specimens of the Panc1shctrl group, we observed an increased expression of Six1 at the invasive edge where EMT plays an important role for tumour invasion. Moreover, we evaluated the expression of CD44 and CD24 in those murine tumour samples to assess the co-expression of EMT markers and surrogate markers associated with a CSC phenotype [27]. Four out of five control tumours were CD44+/CD24+ whereas all Six1-downregulated tumours lost that phenotype and were CD44−/CD24+ (Fig. 3d, e, and f and Additional file 3: Table S2).Fig. 3


Inhibition of Six1 affects tumour invasion and the expression of cancer stem cell markers in pancreatic cancer
Six1 downregulation results in a growth arrest of Panc1 cells in a xenograft model. a Body weight curve of mice. Straight line: Panc-1 tumours with scramble shRNA (Panc1shCtl). Dashed line: Panc-1 tumours with Six1-shRNA (Panc1shSix1). No difference in body weight in both groups. b Tumour growth curve of Panc-1 tumours. Straight line: Panc-1 tumours with scramble shRNA (Panc1shCtl). Dashed line: Panc-1 tumours with Six1-shRNA (Panc1shSix1). c Tumour volume of Panc-1 tumour after resection from xenograft models. Upper panel: Panc-1 tumours with Six1-shRNA (Panc1shSix1). Lower panel: Panc-1 tumours with scramble shRNA (Panc1shCtl). d Representative figures for expression of Six1 in Panc-1 tumours with scramble shRNA (left panel) and tumours with Six1-shRNA (right panel). e,f Representative figures for expression of CD44 e and CD24 f in Panc-1 tumours with scramble shRNA (left panel) and tumours with Six1-shRNA (right panel)
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Fig3: Six1 downregulation results in a growth arrest of Panc1 cells in a xenograft model. a Body weight curve of mice. Straight line: Panc-1 tumours with scramble shRNA (Panc1shCtl). Dashed line: Panc-1 tumours with Six1-shRNA (Panc1shSix1). No difference in body weight in both groups. b Tumour growth curve of Panc-1 tumours. Straight line: Panc-1 tumours with scramble shRNA (Panc1shCtl). Dashed line: Panc-1 tumours with Six1-shRNA (Panc1shSix1). c Tumour volume of Panc-1 tumour after resection from xenograft models. Upper panel: Panc-1 tumours with Six1-shRNA (Panc1shSix1). Lower panel: Panc-1 tumours with scramble shRNA (Panc1shCtl). d Representative figures for expression of Six1 in Panc-1 tumours with scramble shRNA (left panel) and tumours with Six1-shRNA (right panel). e,f Representative figures for expression of CD44 e and CD24 f in Panc-1 tumours with scramble shRNA (left panel) and tumours with Six1-shRNA (right panel)
Mentions: We assessed the effect of Six1 inhibition in Panc1 cells in vivo in a xenograft model. For this analysis, shRNA-transfected Panc1 cells were used, showing a stably decreased expression of Six1. After injection of tumour cells into nude mice (2.5 × 106 cells/mouse), body weight and tumour growth were observed for four weeks (Fig. 3a–c). The body weight did not show any difference between these two groups during the observation time. In the beginning, the average tumour volume was 47.65 mm3 (±19.34) in the control group (Panc1shctrl) and 47.00 mm3 (±15.90) in the group with Six1-shRNA (Panc1shSix1). After two weeks, the tumour volume had increased to 86.78 mm3 (±33.93) in the control group and had declined to 39.84 mm3 (±18.54) in Panc1shSix1group (p = 0.0018). At time of euthanasia, the average tumour volume was 124.13 mm3 (±46.59) in the Panc1shctrl group and 50.22 mm3 (±29.76) in Panc1shSix1 (p = 0.0008). After euthanasia, tumour samples of both groups were immunostained against Six1. As expected, tumours from the Panc1shctrl group showed a higher expression of Six1 than tumours from the Panc1shSix1 group (Fig. 3d). Interestingly, in the tumour specimens of the Panc1shctrl group, we observed an increased expression of Six1 at the invasive edge where EMT plays an important role for tumour invasion. Moreover, we evaluated the expression of CD44 and CD24 in those murine tumour samples to assess the co-expression of EMT markers and surrogate markers associated with a CSC phenotype [27]. Four out of five control tumours were CD44+/CD24+ whereas all Six1-downregulated tumours lost that phenotype and were CD44−/CD24+ (Fig. 3d, e, and f and Additional file 3: Table S2).Fig. 3

View Article: PubMed Central - PubMed

ABSTRACT

Background: Epithelial-to-mesenchymal transition (EMT) and cancer stem cells (CSC) contribute to tumour progression and metastasis. Assessment of transcription factors involved in these two mechanisms can help to identify new targets for an oncological therapy. In this study, we focused on the evaluation of the transcription factor Six1 (Sine oculis 1). This protein is involved in embryologic development and its contribution to carcinogenesis has been described in several studies.

Methods: Immunohistochemistry against Six1 was performed on a tissue microarray containing specimens of primary pancreatic ductal adenocarcinomas (PDAC) of 139 patients. Nuclear and cytoplasmic expression was evaluated and correlated to histopathological parameters. Expression of Six1 was inhibited transiently by siRNA in Panc1 and BxPc3 cells and stably by shRNA in Panc1 cells. Expression analysis of CDH1 and Vimentin mRNA was performed and cell motility was tested in a migration assay. Panc1 cells transfected with Six1 shRNA or scrambled shRNA were injected subcutaneously into nude mice. Tumour growth was observed for four weeks. Afterwards, tumours were stained against Six1, CD24 and CD44.

Results: Six1 was overexpressed in the cytoplasm and cellular nuclei in malignant tissues (p < 0.0001). No correlation to histopathological parameters could be detected. Six1 down-regulation decreased pancreatic cancer cell motility in vitro. CDH1 and vimentin expression was decreased after inhibition of the expression of Six1. Pancreatic tumours with impaired expression of Six1 showed significantly delayed growth and displayed loss of the CD24+/CD44+ phenotype.

Conclusion: We show that Six1 is overexpressed in human PDAC and that its inhibition results in a decreased tumour progression in vitro and in vivo. Therefore, targeting Six1 might be a novel therapeutic approach in patients with pancreatic cancer.

Electronic supplementary material: The online version of this article (doi:10.1186/s12885-017-3225-5) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus