Limits...
Knockdown of CD44 inhibits the invasion and metastasis of hepatocellular carcinoma both in vitro and in vivo by reversing epithelial-mesenchymal transition.

Gao Y, Ruan B, Liu W, Wang J, Yang X, Zhang Z, Li X, Duan J, Zhang F, Ding R, Tao K, Dou K - Oncotarget (2015)

Bottom Line: First, we noticed that CD44 expression was associated with the mesenchymal phenotype in HCC cell lines, and knocking down CD44 with lentivirus-mediated shRNA in HCC cell lines resulted in the mesenchymal-epithelial-transition (MET) and the subsequent impaired migration and invasion in vitro.Moreover, in a metastatic mice model established by tail vein injection of luciferase labelled MHCC97-H cells, we confirmed that CD44 knockdown resulted in the decreased metastasis of HCC cells.Furthermore, we found that the induction of MET by CD44 inhibition might be achieved, at least in part, by repressing the ERK/Snail pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Hepato-Biliary and Pancreto-Splenic Surgery, Xijing Hospital, The Fourth Military Medical University, Xi'an, China.

ABSTRACT
Mounting evidence has shown that induction of epithelial-mesenchymal transition (EMT) contributes to the the expression of CSC (cancer stem cell) markers. However, whether and how CSC markers could be involved in regulating EMT has rarely been reported. CD44, being one of the most commonly used CSC markers in hepatocellular carcinoma (HCC), has been demonstrated to act as a multidomain, transmembrane platform that serves to integrate a wide variety of extracellular signals. Therefore, we determined to seek whether CD44 is necessary for the EMT process in HCC. First, we noticed that CD44 expression was associated with the mesenchymal phenotype in HCC cell lines, and knocking down CD44 with lentivirus-mediated shRNA in HCC cell lines resulted in the mesenchymal-epithelial-transition (MET) and the subsequent impaired migration and invasion in vitro. Moreover, in a metastatic mice model established by tail vein injection of luciferase labelled MHCC97-H cells, we confirmed that CD44 knockdown resulted in the decreased metastasis of HCC cells. Furthermore, we found that the induction of MET by CD44 inhibition might be achieved, at least in part, by repressing the ERK/Snail pathway.

No MeSH data available.


Related in: MedlinePlus

Knockdown of CD44 induced the MET in SMMC-7721 and MHCC97-H cells by repressing the ERK/Snail pathway(A) qRT-PCR analysis of the mRNA expression of transcription factors that predominantly drive EMT in SMMC-7721 and MHCC97-H cells transfected with shRNA of CD44 or NC. (B) Quantification of Western blot showed different phospho-ERK and Snail expression in SMMC-7721 and MHCC97-H cells with or without CD44-knockdown. (C) The expression of CD44, ERK, p-ERK and Snail was detected in metastatic lung samples by immunohistochemical staining and shown with a 400× field.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4480719&req=5

Figure 5: Knockdown of CD44 induced the MET in SMMC-7721 and MHCC97-H cells by repressing the ERK/Snail pathway(A) qRT-PCR analysis of the mRNA expression of transcription factors that predominantly drive EMT in SMMC-7721 and MHCC97-H cells transfected with shRNA of CD44 or NC. (B) Quantification of Western blot showed different phospho-ERK and Snail expression in SMMC-7721 and MHCC97-H cells with or without CD44-knockdown. (C) The expression of CD44, ERK, p-ERK and Snail was detected in metastatic lung samples by immunohistochemical staining and shown with a 400× field.

Mentions: Because the hallmark of the EMT is directly or indirectly modulated by several predominant transcription factors, including Snail, Twist1, ZEB2 and FOXC2 [8-10, 27-29], we searched for the possible transcription factors that are regulated by CD44. Among these screened transcription factors, we found that both the mRNA (Fig. 5A) and protein (Fig. 5B) expression of Snail were markedly decreased in both SMMC-7721 and MHCC97-H cells in the KD group compared to their corresponding NC cells. At the mean time there were no significant or consistent changes of ZEB2, Twist1 and FOXC2 mRNA in SMMC-7721 and MHCC97-H cells (Fig. 5A). Because CD44 has been demonstrated to integrate the signaling of RTKs [23], which activates the Ras/Raf/MEK/ERK signaling cascade to phosphorylate ERK [30, 31], and mounting evidence indicated that activation of ERK positively regulates the expression of Snail [32-35], we investigated whether the knockdown of CD44 repressed Snail by inhibiting ERK phosphorylation. Western blot analysis showed that the phosphorylation of ERK1/2 (Thr202/Tyr204) was strongly inhibited in SMMC-7721-KD and MHCC97-H-KD cells, unlike in SMMC-7721-NC and MHCC97-H-NC cells cultured under normal conditions, whereas the total ERK1/2 remained unchanged (Fig. 5B). Moreover, immunohistochemical analysis of the metastatic nodule in nude mice showed decreased ERK phosphorylation (Thr202/Tyr204) and Snail expression in response to CD44 knockdown (Fig. 5C). Taken together, these data indicated that CD44 knockdown might induce the MET in SMMC-7721 and MHCC97-H cells by inhibiting the ERK/Snail pathway, at least in part.


Knockdown of CD44 inhibits the invasion and metastasis of hepatocellular carcinoma both in vitro and in vivo by reversing epithelial-mesenchymal transition.

Gao Y, Ruan B, Liu W, Wang J, Yang X, Zhang Z, Li X, Duan J, Zhang F, Ding R, Tao K, Dou K - Oncotarget (2015)

Knockdown of CD44 induced the MET in SMMC-7721 and MHCC97-H cells by repressing the ERK/Snail pathway(A) qRT-PCR analysis of the mRNA expression of transcription factors that predominantly drive EMT in SMMC-7721 and MHCC97-H cells transfected with shRNA of CD44 or NC. (B) Quantification of Western blot showed different phospho-ERK and Snail expression in SMMC-7721 and MHCC97-H cells with or without CD44-knockdown. (C) The expression of CD44, ERK, p-ERK and Snail was detected in metastatic lung samples by immunohistochemical staining and shown with a 400× field.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4480719&req=5

Figure 5: Knockdown of CD44 induced the MET in SMMC-7721 and MHCC97-H cells by repressing the ERK/Snail pathway(A) qRT-PCR analysis of the mRNA expression of transcription factors that predominantly drive EMT in SMMC-7721 and MHCC97-H cells transfected with shRNA of CD44 or NC. (B) Quantification of Western blot showed different phospho-ERK and Snail expression in SMMC-7721 and MHCC97-H cells with or without CD44-knockdown. (C) The expression of CD44, ERK, p-ERK and Snail was detected in metastatic lung samples by immunohistochemical staining and shown with a 400× field.
Mentions: Because the hallmark of the EMT is directly or indirectly modulated by several predominant transcription factors, including Snail, Twist1, ZEB2 and FOXC2 [8-10, 27-29], we searched for the possible transcription factors that are regulated by CD44. Among these screened transcription factors, we found that both the mRNA (Fig. 5A) and protein (Fig. 5B) expression of Snail were markedly decreased in both SMMC-7721 and MHCC97-H cells in the KD group compared to their corresponding NC cells. At the mean time there were no significant or consistent changes of ZEB2, Twist1 and FOXC2 mRNA in SMMC-7721 and MHCC97-H cells (Fig. 5A). Because CD44 has been demonstrated to integrate the signaling of RTKs [23], which activates the Ras/Raf/MEK/ERK signaling cascade to phosphorylate ERK [30, 31], and mounting evidence indicated that activation of ERK positively regulates the expression of Snail [32-35], we investigated whether the knockdown of CD44 repressed Snail by inhibiting ERK phosphorylation. Western blot analysis showed that the phosphorylation of ERK1/2 (Thr202/Tyr204) was strongly inhibited in SMMC-7721-KD and MHCC97-H-KD cells, unlike in SMMC-7721-NC and MHCC97-H-NC cells cultured under normal conditions, whereas the total ERK1/2 remained unchanged (Fig. 5B). Moreover, immunohistochemical analysis of the metastatic nodule in nude mice showed decreased ERK phosphorylation (Thr202/Tyr204) and Snail expression in response to CD44 knockdown (Fig. 5C). Taken together, these data indicated that CD44 knockdown might induce the MET in SMMC-7721 and MHCC97-H cells by inhibiting the ERK/Snail pathway, at least in part.

Bottom Line: First, we noticed that CD44 expression was associated with the mesenchymal phenotype in HCC cell lines, and knocking down CD44 with lentivirus-mediated shRNA in HCC cell lines resulted in the mesenchymal-epithelial-transition (MET) and the subsequent impaired migration and invasion in vitro.Moreover, in a metastatic mice model established by tail vein injection of luciferase labelled MHCC97-H cells, we confirmed that CD44 knockdown resulted in the decreased metastasis of HCC cells.Furthermore, we found that the induction of MET by CD44 inhibition might be achieved, at least in part, by repressing the ERK/Snail pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Hepato-Biliary and Pancreto-Splenic Surgery, Xijing Hospital, The Fourth Military Medical University, Xi'an, China.

ABSTRACT
Mounting evidence has shown that induction of epithelial-mesenchymal transition (EMT) contributes to the the expression of CSC (cancer stem cell) markers. However, whether and how CSC markers could be involved in regulating EMT has rarely been reported. CD44, being one of the most commonly used CSC markers in hepatocellular carcinoma (HCC), has been demonstrated to act as a multidomain, transmembrane platform that serves to integrate a wide variety of extracellular signals. Therefore, we determined to seek whether CD44 is necessary for the EMT process in HCC. First, we noticed that CD44 expression was associated with the mesenchymal phenotype in HCC cell lines, and knocking down CD44 with lentivirus-mediated shRNA in HCC cell lines resulted in the mesenchymal-epithelial-transition (MET) and the subsequent impaired migration and invasion in vitro. Moreover, in a metastatic mice model established by tail vein injection of luciferase labelled MHCC97-H cells, we confirmed that CD44 knockdown resulted in the decreased metastasis of HCC cells. Furthermore, we found that the induction of MET by CD44 inhibition might be achieved, at least in part, by repressing the ERK/Snail pathway.

No MeSH data available.


Related in: MedlinePlus