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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 tumorigenesis and metastasis in vivo(A) SK, SKM, P1 and sN1 cells were subcutaneously injected into the flanks of nude mice, respectively, and tumor volumes measured once a week (n = 4 for each group). Tumor growth curves are shown. (B) Six weeks later, tumors were excised and weighed. C–E, Tumors dissected from mice subjected to subcutaneous injection with control and SK-sNEDD4 cells were introduced orthotopically into nude mice and monitored weekly with IVIS. (C) left, Representative IVIS images of mice 5 weeks after tumor implantation (9 mice/group). The metastasis-free survival curves are shown (C, right). Mice were sacrificed 8 weeks after implantation. Livers (the implantation site is highlighted with arrows in panel D) and lungs were excised, fixed and embedded in paraffin. Hematoxylin & Eosin (H&E) staining was performed on liver (E, left) and lung (E, right) tissue sections. N, normal; T, tumor; *P < 0.05; **P < 0.01; ***P < 0.001.
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Figure 4: sNEDD4 promotes tumorigenesis and metastasis in vivo(A) SK, SKM, P1 and sN1 cells were subcutaneously injected into the flanks of nude mice, respectively, and tumor volumes measured once a week (n = 4 for each group). Tumor growth curves are shown. (B) Six weeks later, tumors were excised and weighed. C–E, Tumors dissected from mice subjected to subcutaneous injection with control and SK-sNEDD4 cells were introduced orthotopically into nude mice and monitored weekly with IVIS. (C) left, Representative IVIS images of mice 5 weeks after tumor implantation (9 mice/group). The metastasis-free survival curves are shown (C, right). Mice were sacrificed 8 weeks after implantation. Livers (the implantation site is highlighted with arrows in panel D) and lungs were excised, fixed and embedded in paraffin. Hematoxylin & Eosin (H&E) staining was performed on liver (E, left) and lung (E, right) tissue sections. N, normal; T, tumor; *P < 0.05; **P < 0.01; ***P < 0.001.

Mentions: The subcutaneous model of human HCC in nude mice was used to assess the effects of sNEDD4 on tumorigenesis in vivo. SK, SKM, P1 and sN1 cells were injected subcutaneously into the flanks of male nude mice, and tumor growth curves and endpoint tumor weights obtained, as shown in Figure 4A and 4B. Tumors derived from SKM and SN1 cells grew significantly faster than those from SK and P1 cells, respectively (Figure 4A). Consistent with this, the endpoint tumor weights of SKM and sN1-derived tumors were significantly higher than those of tumors derived from SK and P1, respectively (Figure 4B). Subsequently, the metastatic abilities of control and sNEDD4-overexpressing SK cells were confirmed using an orthotopic mouse model. Metastasis was monitored with in vivo imaging system (IVIS) weekly after implantation of tumor mass. Lung metastatic signals were observed in three out of eight mice implantated with sNEDD4-expressing tumors 3 weeks later. More than half the mice (5/8) harbored lung metastatic signals 5 weeks later. In contrast, lung metastatic signals were not observed in mice in the control group, even at 8 weeks after implantation (Figure 4C, left). Metastasis-free survival of mouse bearing control or SK-sNEDD4 tumor was presented as Kaplan-Meier survival curves (Figure 4C, right). The implanted sNEDD4-expressing tumors grew faster than control tumors and invaded peripheral liver tissues, while a clear rim separating tumors from peripheral liver tissues was observed in control tumors (Figure 4D and 4E left panel). Lung metastases were confirmed via H&E staining of lung tissue sections (Figure 4E, right panel).


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 tumorigenesis and metastasis in vivo(A) SK, SKM, P1 and sN1 cells were subcutaneously injected into the flanks of nude mice, respectively, and tumor volumes measured once a week (n = 4 for each group). Tumor growth curves are shown. (B) Six weeks later, tumors were excised and weighed. C–E, Tumors dissected from mice subjected to subcutaneous injection with control and SK-sNEDD4 cells were introduced orthotopically into nude mice and monitored weekly with IVIS. (C) left, Representative IVIS images of mice 5 weeks after tumor implantation (9 mice/group). The metastasis-free survival curves are shown (C, right). Mice were sacrificed 8 weeks after implantation. Livers (the implantation site is highlighted with arrows in panel D) and lungs were excised, fixed and embedded in paraffin. Hematoxylin & Eosin (H&E) staining was performed on liver (E, left) and lung (E, right) tissue sections. N, normal; T, tumor; *P < 0.05; **P < 0.01; ***P < 0.001.
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Related In: Results  -  Collection

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Figure 4: sNEDD4 promotes tumorigenesis and metastasis in vivo(A) SK, SKM, P1 and sN1 cells were subcutaneously injected into the flanks of nude mice, respectively, and tumor volumes measured once a week (n = 4 for each group). Tumor growth curves are shown. (B) Six weeks later, tumors were excised and weighed. C–E, Tumors dissected from mice subjected to subcutaneous injection with control and SK-sNEDD4 cells were introduced orthotopically into nude mice and monitored weekly with IVIS. (C) left, Representative IVIS images of mice 5 weeks after tumor implantation (9 mice/group). The metastasis-free survival curves are shown (C, right). Mice were sacrificed 8 weeks after implantation. Livers (the implantation site is highlighted with arrows in panel D) and lungs were excised, fixed and embedded in paraffin. Hematoxylin & Eosin (H&E) staining was performed on liver (E, left) and lung (E, right) tissue sections. N, normal; T, tumor; *P < 0.05; **P < 0.01; ***P < 0.001.
Mentions: The subcutaneous model of human HCC in nude mice was used to assess the effects of sNEDD4 on tumorigenesis in vivo. SK, SKM, P1 and sN1 cells were injected subcutaneously into the flanks of male nude mice, and tumor growth curves and endpoint tumor weights obtained, as shown in Figure 4A and 4B. Tumors derived from SKM and SN1 cells grew significantly faster than those from SK and P1 cells, respectively (Figure 4A). Consistent with this, the endpoint tumor weights of SKM and sN1-derived tumors were significantly higher than those of tumors derived from SK and P1, respectively (Figure 4B). Subsequently, the metastatic abilities of control and sNEDD4-overexpressing SK cells were confirmed using an orthotopic mouse model. Metastasis was monitored with in vivo imaging system (IVIS) weekly after implantation of tumor mass. Lung metastatic signals were observed in three out of eight mice implantated with sNEDD4-expressing tumors 3 weeks later. More than half the mice (5/8) harbored lung metastatic signals 5 weeks later. In contrast, lung metastatic signals were not observed in mice in the control group, even at 8 weeks after implantation (Figure 4C, left). Metastasis-free survival of mouse bearing control or SK-sNEDD4 tumor was presented as Kaplan-Meier survival curves (Figure 4C, right). The implanted sNEDD4-expressing tumors grew faster than control tumors and invaded peripheral liver tissues, while a clear rim separating tumors from peripheral liver tissues was observed in control tumors (Figure 4D and 4E left panel). Lung metastases were confirmed via H&E staining of lung tissue sections (Figure 4E, right panel).

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