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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

Generation of potent metastatic cell lines, SKT and SKM, and identifying metastatic-associated gene, sNEDD4(A) Schematic diagram of the experimental procedure. (B) The invasive abilities of SK, SKT, and SKM cells were determined with the Transwell invasion assay. (C) Western blot analysis was used to determined NEDD4 protein expression of SK, SKT, and SKM cells. Three (left; upper, middle, and lower) and two (right; upper and lower) major bands were detected using NEDD4 and NEDD4–1 antibodies in SKM cells, respectively. (D) Schematic diagram of NEDD4 mRNA (Upper). The open box represents the coding region of NEDD4. Classic and predictive ATG sites are labeled with gray bars. The lower schematic diagram represents NEDD4 and sNEDD4 protein structures. (E) NEDD4 mRNA levels were determined via qRT-PCR with primers amplified from two different segments, S1 and S2 (mark in panel D). (F) NEDD4 and sNEDD4 protein levels of SK, SKM and SK cells transiently transfected with empty vector (Vec) or sNEDD4 expression plasmid (sNED). (G)sNEDD4 was detected using Northern blot. SK cells transfected with sNEDD4 expressing plasmid (sNED) serve as a positive control. IB, immunobloting; S1, 366–478 nucleotides; S2, 649–751 nucleotides; ***P < 0.001.
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Figure 1: Generation of potent metastatic cell lines, SKT and SKM, and identifying metastatic-associated gene, sNEDD4(A) Schematic diagram of the experimental procedure. (B) The invasive abilities of SK, SKT, and SKM cells were determined with the Transwell invasion assay. (C) Western blot analysis was used to determined NEDD4 protein expression of SK, SKT, and SKM cells. Three (left; upper, middle, and lower) and two (right; upper and lower) major bands were detected using NEDD4 and NEDD4–1 antibodies in SKM cells, respectively. (D) Schematic diagram of NEDD4 mRNA (Upper). The open box represents the coding region of NEDD4. Classic and predictive ATG sites are labeled with gray bars. The lower schematic diagram represents NEDD4 and sNEDD4 protein structures. (E) NEDD4 mRNA levels were determined via qRT-PCR with primers amplified from two different segments, S1 and S2 (mark in panel D). (F) NEDD4 and sNEDD4 protein levels of SK, SKM and SK cells transiently transfected with empty vector (Vec) or sNEDD4 expression plasmid (sNED). (G)sNEDD4 was detected using Northern blot. SK cells transfected with sNEDD4 expressing plasmid (sNED) serve as a positive control. IB, immunobloting; S1, 366–478 nucleotides; S2, 649–751 nucleotides; ***P < 0.001.

Mentions: The in vitro Transwell assay and in vivo mouse model were employed to select cells with potent metastatic properties (SKT and SKM cells), with the aim of identifying metastasis-regulating genes. Experimental procedures are summarized in Figure 1A. The invasive properties of SK, SKT and SKM cells were determined using the Transwell invasion assay. Our results showed that the invasion abilities of SKT and SKM cells are significantly increased, compared to that of SK parental cells (Figure 1B). However, analysis of proliferation activity revealed no significant differences between the cell lines (data not shown). The potent metastatic cells were successfully generated.


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)

Generation of potent metastatic cell lines, SKT and SKM, and identifying metastatic-associated gene, sNEDD4(A) Schematic diagram of the experimental procedure. (B) The invasive abilities of SK, SKT, and SKM cells were determined with the Transwell invasion assay. (C) Western blot analysis was used to determined NEDD4 protein expression of SK, SKT, and SKM cells. Three (left; upper, middle, and lower) and two (right; upper and lower) major bands were detected using NEDD4 and NEDD4–1 antibodies in SKM cells, respectively. (D) Schematic diagram of NEDD4 mRNA (Upper). The open box represents the coding region of NEDD4. Classic and predictive ATG sites are labeled with gray bars. The lower schematic diagram represents NEDD4 and sNEDD4 protein structures. (E) NEDD4 mRNA levels were determined via qRT-PCR with primers amplified from two different segments, S1 and S2 (mark in panel D). (F) NEDD4 and sNEDD4 protein levels of SK, SKM and SK cells transiently transfected with empty vector (Vec) or sNEDD4 expression plasmid (sNED). (G)sNEDD4 was detected using Northern blot. SK cells transfected with sNEDD4 expressing plasmid (sNED) serve as a positive control. IB, immunobloting; S1, 366–478 nucleotides; S2, 649–751 nucleotides; ***P < 0.001.
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Related In: Results  -  Collection

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Figure 1: Generation of potent metastatic cell lines, SKT and SKM, and identifying metastatic-associated gene, sNEDD4(A) Schematic diagram of the experimental procedure. (B) The invasive abilities of SK, SKT, and SKM cells were determined with the Transwell invasion assay. (C) Western blot analysis was used to determined NEDD4 protein expression of SK, SKT, and SKM cells. Three (left; upper, middle, and lower) and two (right; upper and lower) major bands were detected using NEDD4 and NEDD4–1 antibodies in SKM cells, respectively. (D) Schematic diagram of NEDD4 mRNA (Upper). The open box represents the coding region of NEDD4. Classic and predictive ATG sites are labeled with gray bars. The lower schematic diagram represents NEDD4 and sNEDD4 protein structures. (E) NEDD4 mRNA levels were determined via qRT-PCR with primers amplified from two different segments, S1 and S2 (mark in panel D). (F) NEDD4 and sNEDD4 protein levels of SK, SKM and SK cells transiently transfected with empty vector (Vec) or sNEDD4 expression plasmid (sNED). (G)sNEDD4 was detected using Northern blot. SK cells transfected with sNEDD4 expressing plasmid (sNED) serve as a positive control. IB, immunobloting; S1, 366–478 nucleotides; S2, 649–751 nucleotides; ***P < 0.001.
Mentions: The in vitro Transwell assay and in vivo mouse model were employed to select cells with potent metastatic properties (SKT and SKM cells), with the aim of identifying metastasis-regulating genes. Experimental procedures are summarized in Figure 1A. The invasive properties of SK, SKT and SKM cells were determined using the Transwell invasion assay. Our results showed that the invasion abilities of SKT and SKM cells are significantly increased, compared to that of SK parental cells (Figure 1B). However, analysis of proliferation activity revealed no significant differences between the cell lines (data not shown). The potent metastatic cells were successfully generated.

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