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

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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.


Expression of Six1, CDH1 and vimentin in Panc1 and BxPc3 cells. a Six1 inhibition by shRNA or siRNA decreases Six1 expression in Panc1 cells in comparison to control (scramble shRNA or siRNA). b Six1 inhibition siRNA decreases Six1 expression in BxPc3 cells in comparison to scramble siRNA. (C) CDH1 expression in Panc1 cells is reduced after Six1 downregulation. d CDH1 expression in BcPc3 is decreased after Six1 downregulation. e Vimentin expression in Panc1 cells is not altered by Six1 downregulation. f Vimentin expression in BxPc3 cells is decreased by downregulation of Six1. g Downregulation of Six1 impairs migration of Panc1 cells. h Downregulation of Six1 impairs migration of BxPc3 cells
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Fig2: Expression of Six1, CDH1 and vimentin in Panc1 and BxPc3 cells. a Six1 inhibition by shRNA or siRNA decreases Six1 expression in Panc1 cells in comparison to control (scramble shRNA or siRNA). b Six1 inhibition siRNA decreases Six1 expression in BxPc3 cells in comparison to scramble siRNA. (C) CDH1 expression in Panc1 cells is reduced after Six1 downregulation. d CDH1 expression in BcPc3 is decreased after Six1 downregulation. e Vimentin expression in Panc1 cells is not altered by Six1 downregulation. f Vimentin expression in BxPc3 cells is decreased by downregulation of Six1. g Downregulation of Six1 impairs migration of Panc1 cells. h Downregulation of Six1 impairs migration of BxPc3 cells

Mentions: Expression of Six1 was inhibited transiently by siRNA in Panc1 cells and BxPc3 cells. This resulted in a decreased expression of Six1 mRNA by 86% in Panc1 cells and by 48% in BxPc3 cells in comparison to controls (Fig. 2a, b). Furthermore, Six1 was stably downregulated in Panc1 cells using shRNA. This resulted in a decreased expression of Six1 by 64.5% when compared to control with scramble shRNA (Fig. 2a). Intriguingly, both approaches lead to a decreased transcription of E-Cadherin mRNA in siRNA-transfected (− 53.2%, p = 0.005) and shRNA-transfected (− 85.2%, p < 0.0001) Panc1 cells (Fig. 2c). Likewise, we observed a decreased expression of CDH1 mRNA in BxPc3 cells, when Six1 siRNA was transfected (−30%, p = 0.03) (Fig. 2d). Furthermore, we investigated the expression of vimentin in both cell lines. There was a slight, but not significant decrease in siRNA-transfected and shRNA- Panc1 cells (Fig. 2e). However, BxPC3 transfected with siRNA against Six1 showed a significantly declined expression of vimentin by 58.6% (p = 0.04) (Fig. 2f).Fig. 2


Inhibition of Six1 affects tumour invasion and the expression of cancer stem cell markers in pancreatic cancer
Expression of Six1, CDH1 and vimentin in Panc1 and BxPc3 cells. a Six1 inhibition by shRNA or siRNA decreases Six1 expression in Panc1 cells in comparison to control (scramble shRNA or siRNA). b Six1 inhibition siRNA decreases Six1 expression in BxPc3 cells in comparison to scramble siRNA. (C) CDH1 expression in Panc1 cells is reduced after Six1 downregulation. d CDH1 expression in BcPc3 is decreased after Six1 downregulation. e Vimentin expression in Panc1 cells is not altered by Six1 downregulation. f Vimentin expression in BxPc3 cells is decreased by downregulation of Six1. g Downregulation of Six1 impairs migration of Panc1 cells. h Downregulation of Six1 impairs migration of BxPc3 cells
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Fig2: Expression of Six1, CDH1 and vimentin in Panc1 and BxPc3 cells. a Six1 inhibition by shRNA or siRNA decreases Six1 expression in Panc1 cells in comparison to control (scramble shRNA or siRNA). b Six1 inhibition siRNA decreases Six1 expression in BxPc3 cells in comparison to scramble siRNA. (C) CDH1 expression in Panc1 cells is reduced after Six1 downregulation. d CDH1 expression in BcPc3 is decreased after Six1 downregulation. e Vimentin expression in Panc1 cells is not altered by Six1 downregulation. f Vimentin expression in BxPc3 cells is decreased by downregulation of Six1. g Downregulation of Six1 impairs migration of Panc1 cells. h Downregulation of Six1 impairs migration of BxPc3 cells
Mentions: Expression of Six1 was inhibited transiently by siRNA in Panc1 cells and BxPc3 cells. This resulted in a decreased expression of Six1 mRNA by 86% in Panc1 cells and by 48% in BxPc3 cells in comparison to controls (Fig. 2a, b). Furthermore, Six1 was stably downregulated in Panc1 cells using shRNA. This resulted in a decreased expression of Six1 by 64.5% when compared to control with scramble shRNA (Fig. 2a). Intriguingly, both approaches lead to a decreased transcription of E-Cadherin mRNA in siRNA-transfected (− 53.2%, p = 0.005) and shRNA-transfected (− 85.2%, p < 0.0001) Panc1 cells (Fig. 2c). Likewise, we observed a decreased expression of CDH1 mRNA in BxPc3 cells, when Six1 siRNA was transfected (−30%, p = 0.03) (Fig. 2d). Furthermore, we investigated the expression of vimentin in both cell lines. There was a slight, but not significant decrease in siRNA-transfected and shRNA- Panc1 cells (Fig. 2e). However, BxPC3 transfected with siRNA against Six1 showed a significantly declined expression of vimentin by 58.6% (p = 0.04) (Fig. 2f).Fig. 2

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&nbsp;&lt;&nbsp;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.