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A novel small-form NEDD4 regulates cell invasiveness and apoptosis to promote tumor metastasis.

Liao CJ, Chi HC, Tsai CY, Chen CD, Wu SM, Tseng YH, Lin YH, Chung IH, Chen CY, Lin SL, Huang SF, Huang YH, Lin KH - Oncotarget (2015)

Bottom Line: A small form of Neural precursor cell-expressed developmentally downregulated 4 (sNEDD4) was identified to be overexpressed in SKM cells, which was confirmed as a novel transcript using liquid chromatography-mass spectrometry.In conclusion, sNEDD4 is a novel metastasis-associated gene, which prevents apoptosis under nutrient restriction conditions.The present findings clearly support the prognostic potential of sNEDD4 for HCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Chang-Gung University, Taoyuan, Taiwan 333, Republic of China.

ABSTRACT
Despite numerous investigations on metastasis, the determinants of metastatic processes remain unclear. We aimed to identify the metastasis-associated genes in hepatocellular carcinoma (HCC). Potent metastatic SK-hep-1 (SK) cells, designated 'SKM', were generated using Transwell assay followed by selection in a mouse model. Genes expressed differentially in SKM and SK cells were identified via microarray analyses. A small form of Neural precursor cell-expressed developmentally downregulated 4 (sNEDD4) was identified to be overexpressed in SKM cells, which was confirmed as a novel transcript using liquid chromatography-mass spectrometry. In clinical specimens, sNEDD4 was significantly overexpressed in tumors and serves as a poor prognostic factor for male patients with HCC (P = 0.035). Upon subcutaneous introduction of sNEDD4-overexpressing SK cells into flanks of nude mice, tumors grew faster than those of the control group. Furthermore, sNEDD4-mediated promotion of tumor metastasis was demonstrated in the orthotopic mouse model. Overexpression of sNEDD4 increased the invasive ability of SK cells through upregulation of matrix metalloproteinase 9 and inhibited serum deprivation-induced apoptosis via upregulation of myeloid cell leukemia 1. In conclusion, sNEDD4 is a novel metastasis-associated gene, which prevents apoptosis under nutrient restriction conditions. The present findings clearly support the prognostic potential of sNEDD4 for HCC.

No MeSH data available.


Related in: MedlinePlus

sNEDD4 promotes tumor cell invasion in vitroNEDD4 and sNEDD4 protein levels of cells were determined using Western blot (A–C upper panel). The invasive ability of cells was determined with the Transwell invasion assay (A–C, middle and lower panel). P1 and P2, control stable SK cell lines; sN1 and sN2, stable sNEDD4-overexpressing SK cells; Vec, empty vector control; FL, full-length NEDD4; S, sNEDD4; CDS, NEDD4 shRNA target to coding region; 3′UTR, NEDD4 shRNA target to the 3′-untranslated regions; *P < 0.05; **P < 0.01; ***P < 0.001.
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Figure 3: sNEDD4 promotes tumor cell invasion in vitroNEDD4 and sNEDD4 protein levels of cells were determined using Western blot (A–C upper panel). The invasive ability of cells was determined with the Transwell invasion assay (A–C, middle and lower panel). P1 and P2, control stable SK cell lines; sN1 and sN2, stable sNEDD4-overexpressing SK cells; Vec, empty vector control; FL, full-length NEDD4; S, sNEDD4; CDS, NEDD4 shRNA target to coding region; 3′UTR, NEDD4 shRNA target to the 3′-untranslated regions; *P < 0.05; **P < 0.01; ***P < 0.001.

Mentions: To determine the effect of sNEDD4 on tumor invasion, stable control (P1 and P2) and sNEDD4-overexpressing (sN1 and sN2) SK cell lines were generated (Figure 3A, upper panel). The invasive abilities of stable cells were determined using Transwell invasion analysis (Figure 3A, middle and lower panels). Our results showed that the invasive abilities of sN1 and sN2 cells are significantly increased compared to P1 and P2 cells. However, no significant differences in proliferation activity were observed between these stable cell lines (data not shown). Simultaneously, invasive ability was dramatically increased when cells were transiently transfected with the sNEDD4 expression plasmid. Notably, cells transfected with full-length NEDD4 displayed only a slight increased in invasive ability (Figures 3B and S4). Conversely, invasive ability was decreased in sN1 cells infected with NEDD4-CDS shRNA (TRCN0000007551) expression virus, which eliminate endogenous NEDD4 and exogenous sNEDD4 simultaneously, compared to those infected with LacZ shRNA (TRCN0000231722; Figure 3C). Upon infection of sN1 cells with NEDD4–3′UTR shRNA (TRCN0000272425) expression virus, which suppresses endogenous NEDD4 only, invasive ability was maintained (Figure 3C). Invasive ability, coincident with higher sNEDD4 expression of sN1-shNEDD4–3′UTR cells, was further enhanced, compared with that of sN1-shLacZ cells. This finding may be attributed to the fact that cells expresses more sNEDD4 to compensate for the function of NEDD4 (Figure 3C).


A novel small-form NEDD4 regulates cell invasiveness and apoptosis to promote tumor metastasis.

Liao CJ, Chi HC, Tsai CY, Chen CD, Wu SM, Tseng YH, Lin YH, Chung IH, Chen CY, Lin SL, Huang SF, Huang YH, Lin KH - Oncotarget (2015)

sNEDD4 promotes tumor cell invasion in vitroNEDD4 and sNEDD4 protein levels of cells were determined using Western blot (A–C upper panel). The invasive ability of cells was determined with the Transwell invasion assay (A–C, middle and lower panel). P1 and P2, control stable SK cell lines; sN1 and sN2, stable sNEDD4-overexpressing SK cells; Vec, empty vector control; FL, full-length NEDD4; S, sNEDD4; CDS, NEDD4 shRNA target to coding region; 3′UTR, NEDD4 shRNA target to the 3′-untranslated regions; *P < 0.05; **P < 0.01; ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496221&req=5

Figure 3: sNEDD4 promotes tumor cell invasion in vitroNEDD4 and sNEDD4 protein levels of cells were determined using Western blot (A–C upper panel). The invasive ability of cells was determined with the Transwell invasion assay (A–C, middle and lower panel). P1 and P2, control stable SK cell lines; sN1 and sN2, stable sNEDD4-overexpressing SK cells; Vec, empty vector control; FL, full-length NEDD4; S, sNEDD4; CDS, NEDD4 shRNA target to coding region; 3′UTR, NEDD4 shRNA target to the 3′-untranslated regions; *P < 0.05; **P < 0.01; ***P < 0.001.
Mentions: To determine the effect of sNEDD4 on tumor invasion, stable control (P1 and P2) and sNEDD4-overexpressing (sN1 and sN2) SK cell lines were generated (Figure 3A, upper panel). The invasive abilities of stable cells were determined using Transwell invasion analysis (Figure 3A, middle and lower panels). Our results showed that the invasive abilities of sN1 and sN2 cells are significantly increased compared to P1 and P2 cells. However, no significant differences in proliferation activity were observed between these stable cell lines (data not shown). Simultaneously, invasive ability was dramatically increased when cells were transiently transfected with the sNEDD4 expression plasmid. Notably, cells transfected with full-length NEDD4 displayed only a slight increased in invasive ability (Figures 3B and S4). Conversely, invasive ability was decreased in sN1 cells infected with NEDD4-CDS shRNA (TRCN0000007551) expression virus, which eliminate endogenous NEDD4 and exogenous sNEDD4 simultaneously, compared to those infected with LacZ shRNA (TRCN0000231722; Figure 3C). Upon infection of sN1 cells with NEDD4–3′UTR shRNA (TRCN0000272425) expression virus, which suppresses endogenous NEDD4 only, invasive ability was maintained (Figure 3C). Invasive ability, coincident with higher sNEDD4 expression of sN1-shNEDD4–3′UTR cells, was further enhanced, compared with that of sN1-shLacZ cells. This finding may be attributed to the fact that cells expresses more sNEDD4 to compensate for the function of NEDD4 (Figure 3C).

Bottom Line: A small form of Neural precursor cell-expressed developmentally downregulated 4 (sNEDD4) was identified to be overexpressed in SKM cells, which was confirmed as a novel transcript using liquid chromatography-mass spectrometry.In conclusion, sNEDD4 is a novel metastasis-associated gene, which prevents apoptosis under nutrient restriction conditions.The present findings clearly support the prognostic potential of sNEDD4 for HCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Chang-Gung University, Taoyuan, Taiwan 333, Republic of China.

ABSTRACT
Despite numerous investigations on metastasis, the determinants of metastatic processes remain unclear. We aimed to identify the metastasis-associated genes in hepatocellular carcinoma (HCC). Potent metastatic SK-hep-1 (SK) cells, designated 'SKM', were generated using Transwell assay followed by selection in a mouse model. Genes expressed differentially in SKM and SK cells were identified via microarray analyses. A small form of Neural precursor cell-expressed developmentally downregulated 4 (sNEDD4) was identified to be overexpressed in SKM cells, which was confirmed as a novel transcript using liquid chromatography-mass spectrometry. In clinical specimens, sNEDD4 was significantly overexpressed in tumors and serves as a poor prognostic factor for male patients with HCC (P = 0.035). Upon subcutaneous introduction of sNEDD4-overexpressing SK cells into flanks of nude mice, tumors grew faster than those of the control group. Furthermore, sNEDD4-mediated promotion of tumor metastasis was demonstrated in the orthotopic mouse model. Overexpression of sNEDD4 increased the invasive ability of SK cells through upregulation of matrix metalloproteinase 9 and inhibited serum deprivation-induced apoptosis via upregulation of myeloid cell leukemia 1. In conclusion, sNEDD4 is a novel metastasis-associated gene, which prevents apoptosis under nutrient restriction conditions. The present findings clearly support the prognostic potential of sNEDD4 for HCC.

No MeSH data available.


Related in: MedlinePlus