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SATB1 Promotes Pancreatic Cancer Growth and Invasion Depending on MYC Activation - PubMed

SATB1 Promotes Pancreatic Cancer Growth and Invasion Depending on MYC Activation

Zheng Chen et al. Dig Dis Sci. 2015 Nov.

Abstract

Background: SATB1 plays an important role in human malignant progression, inducing cancer cell proliferation and metastasis by regulating downstream gene expressions. However, little is known about the underlying mechanisms in which SATB1 promotes pancreatic cancer tumorigenesis.

Aims: To investigate SATB1 expression levels and its biological functions in promoting pancreatic cancer growth and invasion.

Methods: SATB1 expression levels were detected in seven human pancreatic cancer cell lines and 16 pairs of normal pancreatic/pancreatic cancer tissues using RT-PCR and western blot. SW1990 or Capan-1 cells stably knockdown (shRNA) or transiently knockdown (siRNA) SATB1 cells, and PANC-1 stably overexpressing SATB1 cells were investigated with MTT, EdU assay, flow cytometry, and transwell invasion assay for cell proliferation and invasion activity. The binding of SATB1 to MYC promoter region was examined using reporter assay. Expression of SATB1 in 68 pancreatic cancer samples was studied by immunohistochemical staining and scoring.

Results: SATB1 was overexpressed in pancreatic cancer tissues samples, showing strong correlation with pancreatic cancer invasion depth and tumor staging. SATB1 induced MYC mRNA and protein expression; promoted pancreatic cancer cell growth; increased cell population in S phase; and enhanced pancreatic cancer cell invasion in vitro. On the other hand, SATB1 knockdown showed opposite effects. Furthermore, MYC blocking in SATB1-overexpressing cells attenuated the promotion of pancreatic cancer cell growth and invasion. Our data also indicated that SATB1 bound to specific promoter region of MYC.

Conclusions: SATB1 is overexpressed in pancreatic cancer, promoting cancer cell proliferation and invasion through the activation of MYC.

Keywords: Cell cycle; Myelocytomatosis viral oncogene; Pancreatic carcinoma; Special AT-rich binding protein 1.

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Figures

Fig. 1
Fig. 1

Overexpression of SATB1 in pancreatic cancer cell lines and tissue samples. a PCR analysis of SATB1 mRNA expression level in seven human pancreatic cancer cell lines. b SATB1 protein expression level in pancreatic cancer cell lines. c Real-time PCR showing SATB1 mRNA expression level in 16 pairs of human pancreatic cancer tissue samples. Horizontal bars indicate the arithmetic mean. Error bars indicate standard deviation (SD). P < 0.05, Mann–Whitney U test. d Western blot analysis of SATB1 protein expression level in 16 pairs of human pancreatic cancer (C) or normal pancreas tissue (N) samples. e Quantification data of panel (d) ***P < 0.001, t test

Fig. 2
Fig. 2

Stably overexpression or knockdown of SATB1 regulates pancreatic cancer cell proliferation. a SATB1 expression level detected by PCR (left panel) and western blot (right panel) in SW1990 stably knockdown SATB1 (SATB1 shRNA) and control (control) cells. b SATB1 expression level detected by PCR (left panel) and western blot (right panel) in PANC-1 stably overexpressing SATB1 (pcDNA SATB1) or control (pcDNA) cells. c MTT cell growth curve analysis of SW1990 stably knockdown SATB1 or control cells. d MTT cell growth curve analysis of PANC-1 stably overexpressing SATB1 or control cells. e Cell cycle distribution analysis of control (left panel) or SW1990 stably knockdown SATB1 cells (middle panel) and quantification data (right panel). *P < 0.05, **P < 0.01, ***P < 0.001, t test

Fig. 3
Fig. 3

Stably overexpression or knockdown of SATB1 regulates pancreatic cancer cells growth and invasion. a EdU assay analysis of SW1990 stably knockdown SATB1 (SATB1 shRNA) or control (control) cells (left panel) and quantification data (right panel). b EdU assay analysis of PANC-1 stably overexpressing SATB1 (pcDNA SATB1) or control (pcDNA) cells (left panel) and quantification data (right panel). c Transwell migration/invasion analysis of SW1990 stably knockdown SATB1 or control cells (left panel) and quantification data (right panel). d Transwell migration/invasion analysis of PANC-1 stably overexpressing SATB1 or control cells (left panel) and quantification data (right panel), ***P < 0.001, t test

Fig. 4
Fig. 4

Transiently knockdown SATB1 with siRNA decreases pancreatic cancer cells proliferation and invasion. a Western blot results showing SATB1 protein levels in PANC-1 (upper panel) or Capan-1 (lower panel) transiently knockdown SATB1 by siRNA or control cells. b, c EdU cell proliferation analysis of SW1990 or Capan-1 transiently knocking down SATB1 or control cells (left panels) with quantification data (right panels), ***P < 0.001, t test. d, e Transwell migration/invasion analysis of SW1990 or Capan-1 knocking down SATB1 or control cells (left panels) with quantification data (right panels), **P < 0.01, ***P < 0.001, t test

Fig. 5
Fig. 5

SATB1 promotes pancreatic cancer proliferation and invasion dependent on MYC and MMP9 induction. a MYC and MMP9 mRNA or protein expression levels detected by qRT-PCR (left panels) and western blot (right panel) in SW1990 stably knockdown SATB1 (SATB1 shRNA) or control (Control) cells. b MYC and MMP9 mRNA or protein expression levels detected by qRT-PCR (left panels) and western blot (right panel) in PANC-1 stably overexpressing SATB1 (pcDNA SATB1) or control (pcDNA) cells. c Western blot analysis of MYC or MMP9 protein expression levels in PANC-1 stably overexpressing SATB1 or control cells with or without MYC siRNA knockdown. d MTT cell growth curve analysis of PANC-1 stably overexpressing SATB1 or control cells with or without MYC siRNA knockdown, **P < 0.01, ***P < 0.001, compared with control cells, t test. e Transwell migration/invasion analysis of PANC-1 stably overexpressing SATB1 or control cells with or without MYC siRNA knockdown (left panel) and quantification data (right panel), P < 0.01, one-way ANOVA

Fig. 6
Fig. 6

Specific SATB1 binding sites localize in MYC promoter region. a Thirteen different lengths of MYC promoter regions were cloned into PGL3-basic reporter plasmid (upper panel). Localization of specific SATB1 binding site in MYC promoter region in pancreatic cancer cells (lower panel). b First round of luciferase MYC promoter reporter assay analysis in R1 to R7 MYC promoter regions in SW1990 stably knockdown SATB1 (SATB1 shRNA) or control (Control) cells. c Second round of MYC promoter reporter assay analysis in R8 to R13 MYC promoter regions in SW1990 stably knockdown SATB1 (SATB1 shRNA) or control (Control) cells. ***P < 0.001, t test

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