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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

CAFs secreted TGFβ1 to activate TGFβ/Smad signaling in bladder cancer cells.A: TGFβ1 in conditional mediums secreted by 5637 (CTRL), NF and CAF cells were quantified by ELISA. B: The mRNA expression levels of TGFβ1 in CAFs, NFs and three bladder cancer cell lines by qRT-PCR using β-actin gene as the normalization control. C: The expression of phosphorylated Smad2 and total Smad2 protein in the CAF-CM treated bladder cancer cell lines by immunoblotting. β-actin protein was used as the loading control. D: The expression levels of TGFβ1 and TGFβRII in bladder cancer cell lines cultured with CAF-CM were detected by qRT-PCR using β-actin gene as the normalization control. ***P < 0.001.
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f4: CAFs secreted TGFβ1 to activate TGFβ/Smad signaling in bladder cancer cells.A: TGFβ1 in conditional mediums secreted by 5637 (CTRL), NF and CAF cells were quantified by ELISA. B: The mRNA expression levels of TGFβ1 in CAFs, NFs and three bladder cancer cell lines by qRT-PCR using β-actin gene as the normalization control. C: The expression of phosphorylated Smad2 and total Smad2 protein in the CAF-CM treated bladder cancer cell lines by immunoblotting. β-actin protein was used as the loading control. D: The expression levels of TGFβ1 and TGFβRII in bladder cancer cell lines cultured with CAF-CM were detected by qRT-PCR using β-actin gene as the normalization control. ***P < 0.001.

Mentions: In order to identify which CAFs-secreting cytokine may induce EMT and invasion of bladder cancer cells, we compared TGFβ1, TGFβ2 and TGFβ3 mRNA levels in NFs versus CAFs, and found that TGFβ1 was the most highly expressed cytokine in CAFs than NFs (Suppl. Fig. 2). We also examined the CAF-CM, NF-CM and condition medium from 5637 cells (CTRL) using a cytokine ELISA and higher level of TGFβ1 was detected in the CAF-CM (328.8 pg/ml) than those in the NF-CM (116.9 pg/ml) and CTRL (12.6 pg/ml; Fig. 4A). ELISA data also confirmed larger amount of TGFβ1 in CMs from another two CAFs, compared with that in their NF-CMs (Suppl. Fig. 3A). qRT-PCR also confirmed that TGFβ1 mRNA transcripts were the most abundant in CAFs, about 2.7, 7.7, 22.6 and 4.3-fold the amount in NFs, 5637, T24 and J82 cells, respectively (Fig. 4B). Downstream targets of TGFβ1 signaling in three bladder cancer cell lines under CAF-CM stimulation were further tested. Western blotting assay showed that Smad2 was phosphorylated but the total Smad2 expression remained unchanged (Fig. 4C), indicating the activation of canonical TGFβ signaling under the CAF-CM treatment.


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)

CAFs secreted TGFβ1 to activate TGFβ/Smad signaling in bladder cancer cells.A: TGFβ1 in conditional mediums secreted by 5637 (CTRL), NF and CAF cells were quantified by ELISA. B: The mRNA expression levels of TGFβ1 in CAFs, NFs and three bladder cancer cell lines by qRT-PCR using β-actin gene as the normalization control. C: The expression of phosphorylated Smad2 and total Smad2 protein in the CAF-CM treated bladder cancer cell lines by immunoblotting. β-actin protein was used as the loading control. D: The expression levels of TGFβ1 and TGFβRII in bladder cancer cell lines cultured with CAF-CM were detected by qRT-PCR using β-actin gene as the normalization control. ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: CAFs secreted TGFβ1 to activate TGFβ/Smad signaling in bladder cancer cells.A: TGFβ1 in conditional mediums secreted by 5637 (CTRL), NF and CAF cells were quantified by ELISA. B: The mRNA expression levels of TGFβ1 in CAFs, NFs and three bladder cancer cell lines by qRT-PCR using β-actin gene as the normalization control. C: The expression of phosphorylated Smad2 and total Smad2 protein in the CAF-CM treated bladder cancer cell lines by immunoblotting. β-actin protein was used as the loading control. D: The expression levels of TGFβ1 and TGFβRII in bladder cancer cell lines cultured with CAF-CM were detected by qRT-PCR using β-actin gene as the normalization control. ***P < 0.001.
Mentions: In order to identify which CAFs-secreting cytokine may induce EMT and invasion of bladder cancer cells, we compared TGFβ1, TGFβ2 and TGFβ3 mRNA levels in NFs versus CAFs, and found that TGFβ1 was the most highly expressed cytokine in CAFs than NFs (Suppl. Fig. 2). We also examined the CAF-CM, NF-CM and condition medium from 5637 cells (CTRL) using a cytokine ELISA and higher level of TGFβ1 was detected in the CAF-CM (328.8 pg/ml) than those in the NF-CM (116.9 pg/ml) and CTRL (12.6 pg/ml; Fig. 4A). ELISA data also confirmed larger amount of TGFβ1 in CMs from another two CAFs, compared with that in their NF-CMs (Suppl. Fig. 3A). qRT-PCR also confirmed that TGFβ1 mRNA transcripts were the most abundant in CAFs, about 2.7, 7.7, 22.6 and 4.3-fold the amount in NFs, 5637, T24 and J82 cells, respectively (Fig. 4B). Downstream targets of TGFβ1 signaling in three bladder cancer cell lines under CAF-CM stimulation were further tested. Western blotting assay showed that Smad2 was phosphorylated but the total Smad2 expression remained unchanged (Fig. 4C), indicating the activation of canonical TGFβ signaling under the CAF-CM treatment.

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