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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

EMT phenotypes were reversed by blocking TGFβ/Smad signaling in the CAF-CM treated bladder cancer cells.A,B: The protein levels of E-cadherin and Vimentin in the CAF-CM treated 5637 and J82 cells upon the blocking of a neutralizing antibody (A) or a TGFβR1 inhibitor, SB-431542, (B) by immunoblotting. C,D: The mRNA levels of mesenchymal markers and EMT-associated transcription factors in the CAF-CM treated 5637 and J82 cells upon the blocking of a neutralizing antibody (C) or a TGFβR1 inhibitor (SB-431542) (D) by qRT-PCR. Cell migration (E) and invasion ability (F) in the CAF-CM treated 5637, T24 and J82 cells upon the blocking of a neutralizing antibody by the Transwell cell migration/invasion assay. *P < 0.05, **P < 0.01, ***P < 0.001.
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f5: EMT phenotypes were reversed by blocking TGFβ/Smad signaling in the CAF-CM treated bladder cancer cells.A,B: The protein levels of E-cadherin and Vimentin in the CAF-CM treated 5637 and J82 cells upon the blocking of a neutralizing antibody (A) or a TGFβR1 inhibitor, SB-431542, (B) by immunoblotting. C,D: The mRNA levels of mesenchymal markers and EMT-associated transcription factors in the CAF-CM treated 5637 and J82 cells upon the blocking of a neutralizing antibody (C) or a TGFβR1 inhibitor (SB-431542) (D) by qRT-PCR. Cell migration (E) and invasion ability (F) in the CAF-CM treated 5637, T24 and J82 cells upon the blocking of a neutralizing antibody by the Transwell cell migration/invasion assay. *P < 0.05, **P < 0.01, ***P < 0.001.

Mentions: Paracrine activation of TGFβ1 by CAF-CM was further confirmed by the TGFβ1 blocking assays using a neutralizing TGFβ1 antibody or a TGFβRI small molecule inhibitor (SB-431542). Western blotting showed both abrogation of TGFβ1 binding to its cognate receptor (Fig. 5A) or inhibition of TGFβ signaling pathway (Fig. 5B) suppressed the CAF-CM-induced Vimentin expression and slightly increased the expression of E-Cadherin in 5637 and J82 cells. The results were confirmed in another two pairs of CAFs/NFs, when blockade of TGFβ1 signaling by SB-431542 (Suppl. Fig. 3B,C). Consistently, TGFβ1 neutralizing antibody or SB-431542 suppressed the CAF-CM effects on expression of the EMT-associated genes, including Vimentin (VIM), Fibronectin (FN1), SNAI1, ZEB1 and ZEB2, at mRNA level (Fig. 5C,D). In addition, the CAF-CM induced cell migration (Fig. 5E) and invasion (Fig. 5F) could also be inhibited by the TGFβ1 neutralizing antibody in 5637, T24 and J82 cells. The inhibitory effects on invasiveness were also confirmed in another two pairs of CAFs/NFs, when blockade of TGFβ1 signaling by SB-431542 (Suppl. Fig. 3D–G). Moreover, the treatment of three UBC cell lines with TGFβ1 for 48 h induced cancer cell invasion (Suppl. Fig. 4A,B) and EMT associated protein levels (Suppl. Fig. 4C), indicating that TGFβ1 alone is sufficient to induce EMT in UBC cell lines tested.


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)

EMT phenotypes were reversed by blocking TGFβ/Smad signaling in the CAF-CM treated bladder cancer cells.A,B: The protein levels of E-cadherin and Vimentin in the CAF-CM treated 5637 and J82 cells upon the blocking of a neutralizing antibody (A) or a TGFβR1 inhibitor, SB-431542, (B) by immunoblotting. C,D: The mRNA levels of mesenchymal markers and EMT-associated transcription factors in the CAF-CM treated 5637 and J82 cells upon the blocking of a neutralizing antibody (C) or a TGFβR1 inhibitor (SB-431542) (D) by qRT-PCR. Cell migration (E) and invasion ability (F) in the CAF-CM treated 5637, T24 and J82 cells upon the blocking of a neutralizing antibody by the Transwell cell migration/invasion assay. *P < 0.05, **P < 0.01, ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: EMT phenotypes were reversed by blocking TGFβ/Smad signaling in the CAF-CM treated bladder cancer cells.A,B: The protein levels of E-cadherin and Vimentin in the CAF-CM treated 5637 and J82 cells upon the blocking of a neutralizing antibody (A) or a TGFβR1 inhibitor, SB-431542, (B) by immunoblotting. C,D: The mRNA levels of mesenchymal markers and EMT-associated transcription factors in the CAF-CM treated 5637 and J82 cells upon the blocking of a neutralizing antibody (C) or a TGFβR1 inhibitor (SB-431542) (D) by qRT-PCR. Cell migration (E) and invasion ability (F) in the CAF-CM treated 5637, T24 and J82 cells upon the blocking of a neutralizing antibody by the Transwell cell migration/invasion assay. *P < 0.05, **P < 0.01, ***P < 0.001.
Mentions: Paracrine activation of TGFβ1 by CAF-CM was further confirmed by the TGFβ1 blocking assays using a neutralizing TGFβ1 antibody or a TGFβRI small molecule inhibitor (SB-431542). Western blotting showed both abrogation of TGFβ1 binding to its cognate receptor (Fig. 5A) or inhibition of TGFβ signaling pathway (Fig. 5B) suppressed the CAF-CM-induced Vimentin expression and slightly increased the expression of E-Cadherin in 5637 and J82 cells. The results were confirmed in another two pairs of CAFs/NFs, when blockade of TGFβ1 signaling by SB-431542 (Suppl. Fig. 3B,C). Consistently, TGFβ1 neutralizing antibody or SB-431542 suppressed the CAF-CM effects on expression of the EMT-associated genes, including Vimentin (VIM), Fibronectin (FN1), SNAI1, ZEB1 and ZEB2, at mRNA level (Fig. 5C,D). In addition, the CAF-CM induced cell migration (Fig. 5E) and invasion (Fig. 5F) could also be inhibited by the TGFβ1 neutralizing antibody in 5637, T24 and J82 cells. The inhibitory effects on invasiveness were also confirmed in another two pairs of CAFs/NFs, when blockade of TGFβ1 signaling by SB-431542 (Suppl. Fig. 3D–G). Moreover, the treatment of three UBC cell lines with TGFβ1 for 48 h induced cancer cell invasion (Suppl. Fig. 4A,B) and EMT associated protein levels (Suppl. Fig. 4C), indicating that TGFβ1 alone is sufficient to induce EMT in UBC cell lines tested.

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