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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 upregulates Mcl-1 to overcome nutrient deficiency-induced apoptosisStable cells were treated in the presence or absence of low-serum conditions (medium with 1% serum) for 48 h, followed by Annexin V-propidium iodide (PI) double staining, and the proportion of apoptotic cells determined via flow cytometry (A) Protein expression patterns of these cells were determined using Western blot (B) (C) P1 and sN1 cells were transiently transfected with control (shLacZ) or Mcl-1 shRNA (shMcl-1) for 48 h, cultured in low-serum conditions for a further 48 h, and Casp3 activation analyzed. (D) qRT-PCR was used to determine Mcl-1 mRNA levels of stable cells. (E) Mcl-1 protein stability was analyzed with the protein degradation assay. (F) Protein levels of sNEDD4, Mcl-1, and active Casp3 of xenograft tumors on the flanks of mice were determined. CHX, cycloheximide; *P < 0.05; **P < 0.01.
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Figure 6: sNEDD4 upregulates Mcl-1 to overcome nutrient deficiency-induced apoptosisStable cells were treated in the presence or absence of low-serum conditions (medium with 1% serum) for 48 h, followed by Annexin V-propidium iodide (PI) double staining, and the proportion of apoptotic cells determined via flow cytometry (A) Protein expression patterns of these cells were determined using Western blot (B) (C) P1 and sN1 cells were transiently transfected with control (shLacZ) or Mcl-1 shRNA (shMcl-1) for 48 h, cultured in low-serum conditions for a further 48 h, and Casp3 activation analyzed. (D) qRT-PCR was used to determine Mcl-1 mRNA levels of stable cells. (E) Mcl-1 protein stability was analyzed with the protein degradation assay. (F) Protein levels of sNEDD4, Mcl-1, and active Casp3 of xenograft tumors on the flanks of mice were determined. CHX, cycloheximide; *P < 0.05; **P < 0.01.

Mentions: Since nutrient deprivation conditions causing cancer cell apoptosis are common to the tumor microenvironment, tolerance for nutrient deficiency in control and sNEDD4-overexpressing cells was determined. After 48 h incubation in low-serum conditions, cells were harvested and apoptosis detected with the FITC Annexin V Apoptosis Detection Kit (BD Pharmingen). As shown in Figure 6A, the proportion of apoptotic cells was significantly lower in sN1 and sN2 cells, compared to P1 and P2 cells. In addition, active caspase 3 (Casp3) was induced significantly in P1 and P2 cells, but only slightly in sN1 and sN2 cells, under low-serum conditions (Figure 6B). Bcl-2 family proteins are known critical regulators of apoptosis. Therefore, expression levels of several Bcl-2 proteins in stable cells were assessed. Our data showed that myeloid cell leukemia 1 (Mcl-1), one of the anti-apoptosis Bcl-2 family proteins, is significantly upregulated in sN1 and sN2 cells, compared to P1 and P2 cells (Figure 6B). In contrast, we observed no significant differences in Bcl-2, Bim and Bid levels among these stable cell lines (data not shown). Knockdown of Mcl-1 abrogated the protective effect of sNEDD4 against apoptosis (Figure 6C, lane 6 and 8). No significant differences in Mcl-1 mRNA levels were evident among the cell lines examined (Figure 6D). The protein stability of Mcl-1 was further determined. As shown in Figure 6E, Mcl-1 was detectable in sN1, but not P1 cells, after 8 h treatment with cycloheximide (CHX), indicating that Mcl-1 is more stable in sN1 than P1 cells. sNEDD4, Mcl-1 and active Casp3 levels in tumors excised from flanks of nude mice were further analyzed (Figure 6F). sNEDD4 was specifically detected in sN1-derived tumors. Mcl-1 was upregulated, and conversely, active Casp3 was downregulated in sN1-derived tumors. This result was consistent with data obtained in vitro (Figure 6B). Inhibiting of apoptosis by sNEDD4 may contribute to tumorigenesis in mice.


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 upregulates Mcl-1 to overcome nutrient deficiency-induced apoptosisStable cells were treated in the presence or absence of low-serum conditions (medium with 1% serum) for 48 h, followed by Annexin V-propidium iodide (PI) double staining, and the proportion of apoptotic cells determined via flow cytometry (A) Protein expression patterns of these cells were determined using Western blot (B) (C) P1 and sN1 cells were transiently transfected with control (shLacZ) or Mcl-1 shRNA (shMcl-1) for 48 h, cultured in low-serum conditions for a further 48 h, and Casp3 activation analyzed. (D) qRT-PCR was used to determine Mcl-1 mRNA levels of stable cells. (E) Mcl-1 protein stability was analyzed with the protein degradation assay. (F) Protein levels of sNEDD4, Mcl-1, and active Casp3 of xenograft tumors on the flanks of mice were determined. CHX, cycloheximide; *P < 0.05; **P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: sNEDD4 upregulates Mcl-1 to overcome nutrient deficiency-induced apoptosisStable cells were treated in the presence or absence of low-serum conditions (medium with 1% serum) for 48 h, followed by Annexin V-propidium iodide (PI) double staining, and the proportion of apoptotic cells determined via flow cytometry (A) Protein expression patterns of these cells were determined using Western blot (B) (C) P1 and sN1 cells were transiently transfected with control (shLacZ) or Mcl-1 shRNA (shMcl-1) for 48 h, cultured in low-serum conditions for a further 48 h, and Casp3 activation analyzed. (D) qRT-PCR was used to determine Mcl-1 mRNA levels of stable cells. (E) Mcl-1 protein stability was analyzed with the protein degradation assay. (F) Protein levels of sNEDD4, Mcl-1, and active Casp3 of xenograft tumors on the flanks of mice were determined. CHX, cycloheximide; *P < 0.05; **P < 0.01.
Mentions: Since nutrient deprivation conditions causing cancer cell apoptosis are common to the tumor microenvironment, tolerance for nutrient deficiency in control and sNEDD4-overexpressing cells was determined. After 48 h incubation in low-serum conditions, cells were harvested and apoptosis detected with the FITC Annexin V Apoptosis Detection Kit (BD Pharmingen). As shown in Figure 6A, the proportion of apoptotic cells was significantly lower in sN1 and sN2 cells, compared to P1 and P2 cells. In addition, active caspase 3 (Casp3) was induced significantly in P1 and P2 cells, but only slightly in sN1 and sN2 cells, under low-serum conditions (Figure 6B). Bcl-2 family proteins are known critical regulators of apoptosis. Therefore, expression levels of several Bcl-2 proteins in stable cells were assessed. Our data showed that myeloid cell leukemia 1 (Mcl-1), one of the anti-apoptosis Bcl-2 family proteins, is significantly upregulated in sN1 and sN2 cells, compared to P1 and P2 cells (Figure 6B). In contrast, we observed no significant differences in Bcl-2, Bim and Bid levels among these stable cell lines (data not shown). Knockdown of Mcl-1 abrogated the protective effect of sNEDD4 against apoptosis (Figure 6C, lane 6 and 8). No significant differences in Mcl-1 mRNA levels were evident among the cell lines examined (Figure 6D). The protein stability of Mcl-1 was further determined. As shown in Figure 6E, Mcl-1 was detectable in sN1, but not P1 cells, after 8 h treatment with cycloheximide (CHX), indicating that Mcl-1 is more stable in sN1 than P1 cells. sNEDD4, Mcl-1 and active Casp3 levels in tumors excised from flanks of nude mice were further analyzed (Figure 6F). sNEDD4 was specifically detected in sN1-derived tumors. Mcl-1 was upregulated, and conversely, active Casp3 was downregulated in sN1-derived tumors. This result was consistent with data obtained in vitro (Figure 6B). Inhibiting of apoptosis by sNEDD4 may contribute to tumorigenesis in mice.

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