Limits...
TGFβ1 secreted by cancer-associated fibroblasts induces epithelial-mesenchymal transition of bladder cancer cells through lncRNA-ZEB2NAT.

Zhuang J, Lu Q, Shen B, Huang X, Shen L, Zheng X, Huang R, Yan J, Guo H - Sci Rep (2015)

Bottom Line: CAF-CM induced the epithelial-mesenchymal transition (EMT) by regulating expression levels of EMT-associated markers in UBC cells.Additionally, inhibition of TGFβ1 signaling decreased the EMT-associated gene expression, and cancer cell invasion.ZEB2NAT depletion reversed CAF-CM-induced EMT and invasion of cancer cells, as well as reduced the ZEB2 protein level.

View Article: PubMed Central - PubMed

Affiliation: Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing, Jiangsu 210008, China.

ABSTRACT
Urinary bladder cancer (UBC) patients at muscle invasive stage have poor clinical outcome, due to high propensity for metastasis. Cancer-associated fibroblasts (CAFs), one of the principal constituents of the tumor stroma, play an important role in tumor development. However, it is unclear whether CAFs from UBC induce cell invasion and which signaling pathway is involved. Herein, we found that conditional medium from UBC CAFs (CAF-CM) enhanced the invasion of UBC cells. CAF-CM induced the epithelial-mesenchymal transition (EMT) by regulating expression levels of EMT-associated markers in UBC cells. Higher concentration of TGFβ1 in CAF-CM, comparing with the CM from adjacent normal fibroblast, led to phosphorylation of Smad2 in UBC cells. Additionally, inhibition of TGFβ1 signaling decreased the EMT-associated gene expression, and cancer cell invasion. Interestingly, a long non-coding RNA, ZEB2NAT, was demonstrated to be essential for this TGFβ1-dependent process. ZEB2NAT depletion reversed CAF-CM-induced EMT and invasion of cancer cells, as well as reduced the ZEB2 protein level. Consistently, TGFβ1 mRNA expression is positively correlated with ZEB2NAT transcript and ZEB2 protein levels in human bladder cancer specimens. Our data revealed a novel mechanism that CAFs induces EMT and invasion of human UBC cells through the TGFβ1-ZEB2NAT-ZEB2 axis.

No MeSH data available.


Related in: MedlinePlus

CAF-CM induced EMT phenotypes in three bladder cancer cell lines.A: The protein expression levels of E-cadherin, Vimentin, ZEB1 and ZEB2 in the CAF-CM treated bladder cancer cell lines by immunoblotting. β-actin protein was used as the loading control. B: The mRNA expression levels of epithelial marker (CDH1, encoding E-Cadherin), mesenchymal markers (VIM and FN1) (B) invasion markers (MMP2 and MMP9) (C) and EMT-associated transcription factors (D) in the CAF-CM treated bladder cancer cell lines by qRT-PCR using β-actin gene as the normalization control. *P < 0.05, **P < 0.01, ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4495469&req=5

f3: CAF-CM induced EMT phenotypes in three bladder cancer cell lines.A: The protein expression levels of E-cadherin, Vimentin, ZEB1 and ZEB2 in the CAF-CM treated bladder cancer cell lines by immunoblotting. β-actin protein was used as the loading control. B: The mRNA expression levels of epithelial marker (CDH1, encoding E-Cadherin), mesenchymal markers (VIM and FN1) (B) invasion markers (MMP2 and MMP9) (C) and EMT-associated transcription factors (D) in the CAF-CM treated bladder cancer cell lines by qRT-PCR using β-actin gene as the normalization control. *P < 0.05, **P < 0.01, ***P < 0.001.

Mentions: EMT has been reported to be related to cancer cell invasion and metastasis. Since morphological changes were observed upon CAF-CM treatment (Fig. 2A and Suppl. Fig. 1C,D), we investigated whether these cell shape changes were EMT by testing the expression levels of EMT-associated genes. Western blotting analysis (Fig. 3A and Suppl. Fig. 1I) showed that in three different epithelial bladder cancer cell lines, the treatment with all of three CAF-CMs led to the decrease of E-Cadherin expression (epithelial cell marker) along with the increase of Vimentin (mesenchymal cell marker), ZEB1 and ZEB2 expression (EMT-associated transcription factor). Quantitative RT-PCR verified that CDH1 gene, encoding for E-Cadherin, was suppressed at mRNA level; whereas two mesenchymal markers (vimentin and fibronectin) were significantly up-regulated in CAF-CM-treated 5637, T24 and J82 cells (Fig. 3B). In addition, invasion related MMP-2 and -9 genes also increased in CAF-CM-treatment group (Fig. 3C). Furthermore, mRNA levels of transcription factors regulating EMT, such as SNAI1, SNAI2, TWIST1, ZEB1 and ZEB2, were also analyzed by quantitative RT-PCR (Fig. 3D). Overexpression of SNAI1 and ZEB1 transcripts were detected in all three bladder cancer cell lines stimulated with CAF-CM. These data implied that EMT was a potential mechanism for cell migration and invasion induced by CAF-CM.


TGFβ1 secreted by cancer-associated fibroblasts induces epithelial-mesenchymal transition of bladder cancer cells through lncRNA-ZEB2NAT.

Zhuang J, Lu Q, Shen B, Huang X, Shen L, Zheng X, Huang R, Yan J, Guo H - Sci Rep (2015)

CAF-CM induced EMT phenotypes in three bladder cancer cell lines.A: The protein expression levels of E-cadherin, Vimentin, ZEB1 and ZEB2 in the CAF-CM treated bladder cancer cell lines by immunoblotting. β-actin protein was used as the loading control. B: The mRNA expression levels of epithelial marker (CDH1, encoding E-Cadherin), mesenchymal markers (VIM and FN1) (B) invasion markers (MMP2 and MMP9) (C) and EMT-associated transcription factors (D) in the CAF-CM treated bladder cancer cell lines by qRT-PCR using β-actin gene as the normalization control. *P < 0.05, **P < 0.01, ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4495469&req=5

f3: CAF-CM induced EMT phenotypes in three bladder cancer cell lines.A: The protein expression levels of E-cadherin, Vimentin, ZEB1 and ZEB2 in the CAF-CM treated bladder cancer cell lines by immunoblotting. β-actin protein was used as the loading control. B: The mRNA expression levels of epithelial marker (CDH1, encoding E-Cadherin), mesenchymal markers (VIM and FN1) (B) invasion markers (MMP2 and MMP9) (C) and EMT-associated transcription factors (D) in the CAF-CM treated bladder cancer cell lines by qRT-PCR using β-actin gene as the normalization control. *P < 0.05, **P < 0.01, ***P < 0.001.
Mentions: EMT has been reported to be related to cancer cell invasion and metastasis. Since morphological changes were observed upon CAF-CM treatment (Fig. 2A and Suppl. Fig. 1C,D), we investigated whether these cell shape changes were EMT by testing the expression levels of EMT-associated genes. Western blotting analysis (Fig. 3A and Suppl. Fig. 1I) showed that in three different epithelial bladder cancer cell lines, the treatment with all of three CAF-CMs led to the decrease of E-Cadherin expression (epithelial cell marker) along with the increase of Vimentin (mesenchymal cell marker), ZEB1 and ZEB2 expression (EMT-associated transcription factor). Quantitative RT-PCR verified that CDH1 gene, encoding for E-Cadherin, was suppressed at mRNA level; whereas two mesenchymal markers (vimentin and fibronectin) were significantly up-regulated in CAF-CM-treated 5637, T24 and J82 cells (Fig. 3B). In addition, invasion related MMP-2 and -9 genes also increased in CAF-CM-treatment group (Fig. 3C). Furthermore, mRNA levels of transcription factors regulating EMT, such as SNAI1, SNAI2, TWIST1, ZEB1 and ZEB2, were also analyzed by quantitative RT-PCR (Fig. 3D). Overexpression of SNAI1 and ZEB1 transcripts were detected in all three bladder cancer cell lines stimulated with CAF-CM. These data implied that EMT was a potential mechanism for cell migration and invasion induced by CAF-CM.

Bottom Line: CAF-CM induced the epithelial-mesenchymal transition (EMT) by regulating expression levels of EMT-associated markers in UBC cells.Additionally, inhibition of TGFβ1 signaling decreased the EMT-associated gene expression, and cancer cell invasion.ZEB2NAT depletion reversed CAF-CM-induced EMT and invasion of cancer cells, as well as reduced the ZEB2 protein level.

View Article: PubMed Central - PubMed

Affiliation: Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing, Jiangsu 210008, China.

ABSTRACT
Urinary bladder cancer (UBC) patients at muscle invasive stage have poor clinical outcome, due to high propensity for metastasis. Cancer-associated fibroblasts (CAFs), one of the principal constituents of the tumor stroma, play an important role in tumor development. However, it is unclear whether CAFs from UBC induce cell invasion and which signaling pathway is involved. Herein, we found that conditional medium from UBC CAFs (CAF-CM) enhanced the invasion of UBC cells. CAF-CM induced the epithelial-mesenchymal transition (EMT) by regulating expression levels of EMT-associated markers in UBC cells. Higher concentration of TGFβ1 in CAF-CM, comparing with the CM from adjacent normal fibroblast, led to phosphorylation of Smad2 in UBC cells. Additionally, inhibition of TGFβ1 signaling decreased the EMT-associated gene expression, and cancer cell invasion. Interestingly, a long non-coding RNA, ZEB2NAT, was demonstrated to be essential for this TGFβ1-dependent process. ZEB2NAT depletion reversed CAF-CM-induced EMT and invasion of cancer cells, as well as reduced the ZEB2 protein level. Consistently, TGFβ1 mRNA expression is positively correlated with ZEB2NAT transcript and ZEB2 protein levels in human bladder cancer specimens. Our data revealed a novel mechanism that CAFs induces EMT and invasion of human UBC cells through the TGFβ1-ZEB2NAT-ZEB2 axis.

No MeSH data available.


Related in: MedlinePlus