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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

Characterization of primary cultured NFs and CAFs.A: The mRNA expression levels of CAF-specific genes, including FAP, FSP1, ACTA2 and CD90, in 5637 cells (an epithelial cell control), NFs and CAFs by qRT-PCR using β-actin gene as the normalization control. B: The protein expression levels of E-Cadherein, Vimentin and α-SMA in 5637 cells, NFs and CAFs were detected by immunoblotting. C: Immunofluorescence staining showed the subcellular location and the expression of E-cadherin, Vimentin and α-SMA in 5637 cells, NFs and CAFs. Scale bar, 10 μM.
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f1: Characterization of primary cultured NFs and CAFs.A: The mRNA expression levels of CAF-specific genes, including FAP, FSP1, ACTA2 and CD90, in 5637 cells (an epithelial cell control), NFs and CAFs by qRT-PCR using β-actin gene as the normalization control. B: The protein expression levels of E-Cadherein, Vimentin and α-SMA in 5637 cells, NFs and CAFs were detected by immunoblotting. C: Immunofluorescence staining showed the subcellular location and the expression of E-cadherin, Vimentin and α-SMA in 5637 cells, NFs and CAFs. Scale bar, 10 μM.

Mentions: The CAFs and NFs were isolated from three bladder tumor tissues and adjacent normal bladder mucosae. In order to test the purity of CAFs and NFs, we examined fibroblast biomarkers in these cells. As shown in Fig. 1A and Suppl. Fig. 1A, mRNA expression levels of four CAF-specific genes, including fibroblast activation protein (FAP), fibroblast specific protein 1 (FSP1), alpha-smooth muscle actin (ACTA2) and CD90, were significantly increased in CAFs, compared to NFs and UBC 5637 cells (an epithelial cell control). Western blotting assay showed that: 1) epithelial cell marker (E-cadherin) was only detected in 5637 bladder cancer cells; 2) mesenchymal cell marker (Vimentin) was highly expressed in both NFs and CAFs; and 3) myofibroblast marker (α-SMA) was overexpressed only in CAFs (Fig. 1B and Suppl. Fig. 1B). Immunocytochemistry staining further confirmed that primary cultured fibroblast populations (NFs and CAFs) only express Vimentin, but not E-Cadherin. α-SMA expression was higher in CAFs than NF and 5637 cells (Fig. 1C). Altogether, these data indicated that we successfully isolated CAFs with high purity from bladder cancer specimen.


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)

Characterization of primary cultured NFs and CAFs.A: The mRNA expression levels of CAF-specific genes, including FAP, FSP1, ACTA2 and CD90, in 5637 cells (an epithelial cell control), NFs and CAFs by qRT-PCR using β-actin gene as the normalization control. B: The protein expression levels of E-Cadherein, Vimentin and α-SMA in 5637 cells, NFs and CAFs were detected by immunoblotting. C: Immunofluorescence staining showed the subcellular location and the expression of E-cadherin, Vimentin and α-SMA in 5637 cells, NFs and CAFs. Scale bar, 10 μM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Characterization of primary cultured NFs and CAFs.A: The mRNA expression levels of CAF-specific genes, including FAP, FSP1, ACTA2 and CD90, in 5637 cells (an epithelial cell control), NFs and CAFs by qRT-PCR using β-actin gene as the normalization control. B: The protein expression levels of E-Cadherein, Vimentin and α-SMA in 5637 cells, NFs and CAFs were detected by immunoblotting. C: Immunofluorescence staining showed the subcellular location and the expression of E-cadherin, Vimentin and α-SMA in 5637 cells, NFs and CAFs. Scale bar, 10 μM.
Mentions: The CAFs and NFs were isolated from three bladder tumor tissues and adjacent normal bladder mucosae. In order to test the purity of CAFs and NFs, we examined fibroblast biomarkers in these cells. As shown in Fig. 1A and Suppl. Fig. 1A, mRNA expression levels of four CAF-specific genes, including fibroblast activation protein (FAP), fibroblast specific protein 1 (FSP1), alpha-smooth muscle actin (ACTA2) and CD90, were significantly increased in CAFs, compared to NFs and UBC 5637 cells (an epithelial cell control). Western blotting assay showed that: 1) epithelial cell marker (E-cadherin) was only detected in 5637 bladder cancer cells; 2) mesenchymal cell marker (Vimentin) was highly expressed in both NFs and CAFs; and 3) myofibroblast marker (α-SMA) was overexpressed only in CAFs (Fig. 1B and Suppl. Fig. 1B). Immunocytochemistry staining further confirmed that primary cultured fibroblast populations (NFs and CAFs) only express Vimentin, but not E-Cadherin. α-SMA expression was higher in CAFs than NF and 5637 cells (Fig. 1C). Altogether, these data indicated that we successfully isolated CAFs with high purity from bladder cancer specimen.

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