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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-Expression in the patient cohort. Staining against Six1 was performed and Six1 expression was determined in cytoplasm and cell nucleus on a tissue microarray including human samples of patients with pancreatic cancer. a Negative Six1 expression in cytoplasm and nucleus b Negative Six1 expression in cytoplasm and positive nucleus staining c Weak Six1 expression in cytoplasm without nucleus staining. d Weak Six1 expression in cytoplasm and positive nucleus staining. e Strong Six1 expression in cytoplasm and negative nucleus staining. Annotations above the panel rows indicate the magnification scale of the figures: first and third row: 40× magnification. Second and fourth row: 100× magnification
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Fig1: Six1-Expression in the patient cohort. Staining against Six1 was performed and Six1 expression was determined in cytoplasm and cell nucleus on a tissue microarray including human samples of patients with pancreatic cancer. a Negative Six1 expression in cytoplasm and nucleus b Negative Six1 expression in cytoplasm and positive nucleus staining c Weak Six1 expression in cytoplasm without nucleus staining. d Weak Six1 expression in cytoplasm and positive nucleus staining. e Strong Six1 expression in cytoplasm and negative nucleus staining. Annotations above the panel rows indicate the magnification scale of the figures: first and third row: 40× magnification. Second and fourth row: 100× magnification

Mentions: We assessed Six1 expression in tissue samples derived from 139 patients with primary PDAC. In 137 out of 139 patients, cancer tissue could be evaluated and from 105 out of 139 patients, normal pancreatic parenchyma could be assessed. Evaluation was performed separately for Six1 expression in cytoplasm and nuclei, respectively (Fig. 1). 63.5 of the malignant specimens showed an expression of Six1 in the cytoplasm, whereas only 13.3% cases of benign tissue were positive for Six1 expression in the cytoplasm (p < 0.0001). In detail, 50 malignant tumours were negative, 66 PDAC samples showed a weak expression of Six1 and 21 tumour specimens had a high expression of Six1. In contrast, 91 specimens representing benign tissue were negative and only 14 samples displayed a weak expression of Six1 (Table 1). A positive nuclear expression of Six1 was observed in 40.8% (± 4.2) of cancer cells (0 = 81 cases; 1 = 31 cases; 2 = 17 cases, 3 = 4 cases, 4 = 4 cases). On the contrary, only 11.4% (± 3.1) of benign pancreatic tissue specimens displayed a positive expression of Six1 in the nucleus (0 = 93 cases; 1 = 10 cases, 2 = 2 cases). Further analysis between the expression of Six1 (cytoplasm and nucleus) and clinical and histopathological data revealed no significant association of those parameters: tumour stage (p = 1.00 and 0.40, respectively), lymph node status (0.29 and 0.48, respectively), tumour grading (0.95 and 0.19, respectively) (Table 1 and Additional file 1: Table S1).Fig. 1


Inhibition of Six1 affects tumour invasion and the expression of cancer stem cell markers in pancreatic cancer
Six1-Expression in the patient cohort. Staining against Six1 was performed and Six1 expression was determined in cytoplasm and cell nucleus on a tissue microarray including human samples of patients with pancreatic cancer. a Negative Six1 expression in cytoplasm and nucleus b Negative Six1 expression in cytoplasm and positive nucleus staining c Weak Six1 expression in cytoplasm without nucleus staining. d Weak Six1 expression in cytoplasm and positive nucleus staining. e Strong Six1 expression in cytoplasm and negative nucleus staining. Annotations above the panel rows indicate the magnification scale of the figures: first and third row: 40× magnification. Second and fourth row: 100× magnification
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5383957&req=5

Fig1: Six1-Expression in the patient cohort. Staining against Six1 was performed and Six1 expression was determined in cytoplasm and cell nucleus on a tissue microarray including human samples of patients with pancreatic cancer. a Negative Six1 expression in cytoplasm and nucleus b Negative Six1 expression in cytoplasm and positive nucleus staining c Weak Six1 expression in cytoplasm without nucleus staining. d Weak Six1 expression in cytoplasm and positive nucleus staining. e Strong Six1 expression in cytoplasm and negative nucleus staining. Annotations above the panel rows indicate the magnification scale of the figures: first and third row: 40× magnification. Second and fourth row: 100× magnification
Mentions: We assessed Six1 expression in tissue samples derived from 139 patients with primary PDAC. In 137 out of 139 patients, cancer tissue could be evaluated and from 105 out of 139 patients, normal pancreatic parenchyma could be assessed. Evaluation was performed separately for Six1 expression in cytoplasm and nuclei, respectively (Fig. 1). 63.5 of the malignant specimens showed an expression of Six1 in the cytoplasm, whereas only 13.3% cases of benign tissue were positive for Six1 expression in the cytoplasm (p < 0.0001). In detail, 50 malignant tumours were negative, 66 PDAC samples showed a weak expression of Six1 and 21 tumour specimens had a high expression of Six1. In contrast, 91 specimens representing benign tissue were negative and only 14 samples displayed a weak expression of Six1 (Table 1). A positive nuclear expression of Six1 was observed in 40.8% (± 4.2) of cancer cells (0 = 81 cases; 1 = 31 cases; 2 = 17 cases, 3 = 4 cases, 4 = 4 cases). On the contrary, only 11.4% (± 3.1) of benign pancreatic tissue specimens displayed a positive expression of Six1 in the nucleus (0 = 93 cases; 1 = 10 cases, 2 = 2 cases). Further analysis between the expression of Six1 (cytoplasm and nucleus) and clinical and histopathological data revealed no significant association of those parameters: tumour stage (p = 1.00 and 0.40, respectively), lymph node status (0.29 and 0.48, respectively), tumour grading (0.95 and 0.19, respectively) (Table 1 and Additional file 1: Table S1).Fig. 1

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.


Related in: MedlinePlus