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Flotillin-2 promotes nasopharyngeal carcinoma metastasis and is necessary for the epithelial-mesenchymal transition induced by transforming growth factor-β.

Zhao L, Lin L, Pan C, Shi M, Liao Y, Bin J, Liao W - Oncotarget (2015)

Bottom Line: In this study, we found that Flot2 expression level positively correlated with the cancer stage in NPC tissues.In NPC cells, silencing Flot2 reversed the metastatic effect induced by TGF-β.Moreover, TGF-β-induced Src phosphorylation was significantly inhibited by Flot2 knocking down.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Nanfang Hospital, Southern Medical University, Guangzhou Guangdong 510515, China.

ABSTRACT
Transforming growth factor-β (TGF-β) promotes cancer metastasis via the epithelial-mesenchymal transition (EMT) but the underlying mechanisms in nasopharyngeal carcinoma (NPC) remain unclear. Flotillin-2 (Flot2), a specialized lipid raft domain in cellular membrane, was reported to promote cancer metastasis. Recently, in neuropathy, it was also suggested that Flot2 was involved in Src activation, which is known as the downstream signal of TGF-β. Therefore, we intended to find out the relationship between Flot2 and TGF-β in the process of nasopharyngeal carcinoma (NPC) metastasis. In this study, we found that Flot2 expression level positively correlated with the cancer stage in NPC tissues. Elevated Flot2 in tumor tissue was an independent prognostic marker, and higher Flot2 expression level showed shorter overall survival time in 181 NPC patients. In NPC cells, silencing Flot2 reversed the metastatic effect induced by TGF-β. Moreover, TGF-β-induced Src phosphorylation was significantly inhibited by Flot2 knocking down. As the consequence of Flot2 inhibition, the expression of the epithelial biomarker E-cadherin was upregulated, while the mesenchymal marker vimentin and signaling transducer β-catenin was suppressed. In conclusions, Flot2 is an indispensable member for TGF-β signaling, which is essential for the EMT process in NPC metastasis. Suppressing Flot2 may be a novel way against TGF-β-induced EMT.

No MeSH data available.


Related in: MedlinePlus

TGF-β-induced EMT was reversed by Flot2 silencing(A) TGF-β-induced CNE-1 NPC cell scattering and morphologic changes were reversed by siFlot2. Scale bar = 50 μm. (B) Wound-healing assay and (C) Transwell assay of NPC cells treated with TGF-β or siFlot2. (D) TGF-β-induced EMT maker (E-cadherin, vimentin and β-catenin) alterations and Src phosphorylation was reversed by siFlot2. (E) Immunofluorescence staining showed the E-cadherin and vimentin expressions changed by TGF-β and siFlot2 in CNE-1 cells. # P < 0.01, + P < 0.001.
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Figure 6: TGF-β-induced EMT was reversed by Flot2 silencing(A) TGF-β-induced CNE-1 NPC cell scattering and morphologic changes were reversed by siFlot2. Scale bar = 50 μm. (B) Wound-healing assay and (C) Transwell assay of NPC cells treated with TGF-β or siFlot2. (D) TGF-β-induced EMT maker (E-cadherin, vimentin and β-catenin) alterations and Src phosphorylation was reversed by siFlot2. (E) Immunofluorescence staining showed the E-cadherin and vimentin expressions changed by TGF-β and siFlot2 in CNE-1 cells. # P < 0.01, + P < 0.001.

Mentions: It was recently reported that Flot2 kept Src activation in the rat optic nerve [20]. Hence, we then asked whether Flot2 was required in TGF-β signaling in human NPC cells. When siFlot2-#1 was added into the TGF-β cultured CNE-1 cells, cell scattering was dramatically suppressed, and cells were kept in colonic growth as the control and siFlot2-#1 groups (Figure 6A). We also noticed that TGF-β-induced pseudopod protrusions were retrograded by siFlot2 (Figure 6A). In the wound-healing assay, despite the rapid healing of the scar area promoted by TGF-β, cell migratory rate was significantly suppressed by Flot2 silencing (Figure 6B). In the transwell assay, silencing Flot2 under TGF-β stimulation suppressed it to the baseline level (Figure 6C). These suggested that Flot2 was essential for TGF-β-induced NPC cell metastasis. Then, we wanted to confirm the protein changes via western blot and immunofluorescence staining. In western blot, silencing Flot2 under TGF-β stimulation significantly inhibited Src phosphorylation, yet total Src expression was not influenced. As a result, E-cadherin, vimentin and β-catenin remained at the baseline level that similar to the TGF-β free control of CNE-1 and 6–10B cells (Figure 6D). As shown under the confocal fluorescence microscope, TGF-β stimulation decreased E-cadherin expression and increased vimentin expression in CNE-1 cells, while silencing Flot2 kept E-cadherin and suppressed vimentin expression under TGF-β stimulation. No obvious difference was found between the group of control and silencing Flot2 alone (Figure 6E). Finally, we conclude from these results that Flot2 was, though not sufficient, yet necessary and essential for the TGF-β-induced EMT signal transduction.


Flotillin-2 promotes nasopharyngeal carcinoma metastasis and is necessary for the epithelial-mesenchymal transition induced by transforming growth factor-β.

Zhao L, Lin L, Pan C, Shi M, Liao Y, Bin J, Liao W - Oncotarget (2015)

TGF-β-induced EMT was reversed by Flot2 silencing(A) TGF-β-induced CNE-1 NPC cell scattering and morphologic changes were reversed by siFlot2. Scale bar = 50 μm. (B) Wound-healing assay and (C) Transwell assay of NPC cells treated with TGF-β or siFlot2. (D) TGF-β-induced EMT maker (E-cadherin, vimentin and β-catenin) alterations and Src phosphorylation was reversed by siFlot2. (E) Immunofluorescence staining showed the E-cadherin and vimentin expressions changed by TGF-β and siFlot2 in CNE-1 cells. # P < 0.01, + P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496397&req=5

Figure 6: TGF-β-induced EMT was reversed by Flot2 silencing(A) TGF-β-induced CNE-1 NPC cell scattering and morphologic changes were reversed by siFlot2. Scale bar = 50 μm. (B) Wound-healing assay and (C) Transwell assay of NPC cells treated with TGF-β or siFlot2. (D) TGF-β-induced EMT maker (E-cadherin, vimentin and β-catenin) alterations and Src phosphorylation was reversed by siFlot2. (E) Immunofluorescence staining showed the E-cadherin and vimentin expressions changed by TGF-β and siFlot2 in CNE-1 cells. # P < 0.01, + P < 0.001.
Mentions: It was recently reported that Flot2 kept Src activation in the rat optic nerve [20]. Hence, we then asked whether Flot2 was required in TGF-β signaling in human NPC cells. When siFlot2-#1 was added into the TGF-β cultured CNE-1 cells, cell scattering was dramatically suppressed, and cells were kept in colonic growth as the control and siFlot2-#1 groups (Figure 6A). We also noticed that TGF-β-induced pseudopod protrusions were retrograded by siFlot2 (Figure 6A). In the wound-healing assay, despite the rapid healing of the scar area promoted by TGF-β, cell migratory rate was significantly suppressed by Flot2 silencing (Figure 6B). In the transwell assay, silencing Flot2 under TGF-β stimulation suppressed it to the baseline level (Figure 6C). These suggested that Flot2 was essential for TGF-β-induced NPC cell metastasis. Then, we wanted to confirm the protein changes via western blot and immunofluorescence staining. In western blot, silencing Flot2 under TGF-β stimulation significantly inhibited Src phosphorylation, yet total Src expression was not influenced. As a result, E-cadherin, vimentin and β-catenin remained at the baseline level that similar to the TGF-β free control of CNE-1 and 6–10B cells (Figure 6D). As shown under the confocal fluorescence microscope, TGF-β stimulation decreased E-cadherin expression and increased vimentin expression in CNE-1 cells, while silencing Flot2 kept E-cadherin and suppressed vimentin expression under TGF-β stimulation. No obvious difference was found between the group of control and silencing Flot2 alone (Figure 6E). Finally, we conclude from these results that Flot2 was, though not sufficient, yet necessary and essential for the TGF-β-induced EMT signal transduction.

Bottom Line: In this study, we found that Flot2 expression level positively correlated with the cancer stage in NPC tissues.In NPC cells, silencing Flot2 reversed the metastatic effect induced by TGF-β.Moreover, TGF-β-induced Src phosphorylation was significantly inhibited by Flot2 knocking down.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Nanfang Hospital, Southern Medical University, Guangzhou Guangdong 510515, China.

ABSTRACT
Transforming growth factor-β (TGF-β) promotes cancer metastasis via the epithelial-mesenchymal transition (EMT) but the underlying mechanisms in nasopharyngeal carcinoma (NPC) remain unclear. Flotillin-2 (Flot2), a specialized lipid raft domain in cellular membrane, was reported to promote cancer metastasis. Recently, in neuropathy, it was also suggested that Flot2 was involved in Src activation, which is known as the downstream signal of TGF-β. Therefore, we intended to find out the relationship between Flot2 and TGF-β in the process of nasopharyngeal carcinoma (NPC) metastasis. In this study, we found that Flot2 expression level positively correlated with the cancer stage in NPC tissues. Elevated Flot2 in tumor tissue was an independent prognostic marker, and higher Flot2 expression level showed shorter overall survival time in 181 NPC patients. In NPC cells, silencing Flot2 reversed the metastatic effect induced by TGF-β. Moreover, TGF-β-induced Src phosphorylation was significantly inhibited by Flot2 knocking down. As the consequence of Flot2 inhibition, the expression of the epithelial biomarker E-cadherin was upregulated, while the mesenchymal marker vimentin and signaling transducer β-catenin was suppressed. In conclusions, Flot2 is an indispensable member for TGF-β signaling, which is essential for the EMT process in NPC metastasis. Suppressing Flot2 may be a novel way against TGF-β-induced EMT.

No MeSH data available.


Related in: MedlinePlus