Limits...
High Skp2/Low p57(Kip2) Expression is Associated with Poor Prognosis in Human Breast Carcinoma.

Yang C, Nan H, Ma J, Jiang L, Guo Q, Han L, Zhang Y, Nan K, Guo H - Breast Cancer (Auckl) (2015)

Bottom Line: Downregulation of p57(Kip2) is involved in tumor progression, and S-phase kinase-associated protein 2 (Skp2) is an E3 ligase that regulates a variety of cell cycle proteins.In addition, univariate analysis showed that Skp2, p57(Kip2), histological grade, lymph node metastasis, and estrogen and progesterone receptors (ER and PR) were all associated with DFS, and multivariate analysis revealed that lymph node metastasis and Skp2 were independent prognostic biomarkers.Half-life and immunoprecipitation (IP) experiments indicated that Skp2 directly interacts with p57(Kip2) and promotes its degradation, rather than its mutant p57(Kip2) (T310A).

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, First Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an, Shannxi, P. R. China.

ABSTRACT
Downregulation of p57(Kip2) is involved in tumor progression, and S-phase kinase-associated protein 2 (Skp2) is an E3 ligase that regulates a variety of cell cycle proteins. However, the prognostic value of p57(Kip2) and its correlation with Skp2 in breast cancer have not been fully elucidated. Here we report our study on the expression of p57(Kip2) and Skp2 in 102 breast cancer patients by immunohistochemistry, and analysis of clinicopathologic parameters in relation to patient prognosis. The expression of p57(Kip2) was negatively associated with Skp2 expression in breast cancer (r = -0.26, P = 0.009). Kaplan-Meier analysis indicated that both high Skp2 and low p57(Kip2) correlated with poor disease-free survival (DFS) (P = 0.05), and a group with the combination of high Skp2/low p57(Kip2) demonstrated even worse DFS (log-rank = 21.118, P < 0.001). In addition, univariate analysis showed that Skp2, p57(Kip2), histological grade, lymph node metastasis, and estrogen and progesterone receptors (ER and PR) were all associated with DFS, and multivariate analysis revealed that lymph node metastasis and Skp2 were independent prognostic biomarkers. The correlation between p57 and Skp2 was further demonstrated in multiple breast cancer cell lines and cell cycle phases. Half-life and immunoprecipitation (IP) experiments indicated that Skp2 directly interacts with p57(Kip2) and promotes its degradation, rather than its mutant p57(Kip2) (T310A). Overall, our findings demonstrate that Skp2 directly degrades p57(Kip2), and an inverse correlation between these proteins (high skp2/low p57(Kip2)) is associated with poor prognosis in breast cancer. Thus, our results indicate a combined prognostic value of these markers in breast cancer diagnosis and treatment.

No MeSH data available.


Related in: MedlinePlus

Association of Skp2 with p57Kip2 degradation in breast cancer cell lines. (A) After transfection with Skp2 siRNA or control for 48 hours, T47D cells were incubated for the indicated times with CHX (20 μg/mL). Cell lysates were then subjected to immunoblot analysis with antibodys to p57Kip2 or Skp2. (B) Cells were transfected with HA-Skp2 and Flag-p57 (WT) or Flag-p57 (T310A) for 48 hours and then incubated with 10 μM MG132 for 4 hours. After that, cell extracts were immunoprecipitated with an antibody against Flag and analyzed by immunoblotting. (C) After transfection with HA-skp2 and Flag-p57 (WT) or Flag-p57 (T310A) for 48 hours, cells were incubated for the indicated times with CHX (20 μg/mL). Cell lysates were then used for immunoblot analysis with antibodies to Flag or HA. β-Actin was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4525793&req=5

f4-bcbcr-suppl.1-2015-013: Association of Skp2 with p57Kip2 degradation in breast cancer cell lines. (A) After transfection with Skp2 siRNA or control for 48 hours, T47D cells were incubated for the indicated times with CHX (20 μg/mL). Cell lysates were then subjected to immunoblot analysis with antibodys to p57Kip2 or Skp2. (B) Cells were transfected with HA-Skp2 and Flag-p57 (WT) or Flag-p57 (T310A) for 48 hours and then incubated with 10 μM MG132 for 4 hours. After that, cell extracts were immunoprecipitated with an antibody against Flag and analyzed by immunoblotting. (C) After transfection with HA-skp2 and Flag-p57 (WT) or Flag-p57 (T310A) for 48 hours, cells were incubated for the indicated times with CHX (20 μg/mL). Cell lysates were then used for immunoblot analysis with antibodies to Flag or HA. β-Actin was used as a loading control.

Mentions: We hypothesized that Skp2 regulates the stability of p57Kip2 in breast cancer. T47D breast cancer cells were transfected with siRNA-Skp2 or control, and then incubated with the protein synthesis inhibitor CHX (cycloheximide, 20 μg/mL) for 0, 1, 2, and 4 hours. After exposure to CHX, T47D cells exhibited an almost complete loss of p57Kip2 within two hours. The degradation of endogenous p57Kip2 was much slower in Skp2-knockdown T47D cells compared with the control cells (Fig. 4A). These results indicate that knockdown of Skp2 inhibits the degradation of p57Kip2 in T47D cells.


High Skp2/Low p57(Kip2) Expression is Associated with Poor Prognosis in Human Breast Carcinoma.

Yang C, Nan H, Ma J, Jiang L, Guo Q, Han L, Zhang Y, Nan K, Guo H - Breast Cancer (Auckl) (2015)

Association of Skp2 with p57Kip2 degradation in breast cancer cell lines. (A) After transfection with Skp2 siRNA or control for 48 hours, T47D cells were incubated for the indicated times with CHX (20 μg/mL). Cell lysates were then subjected to immunoblot analysis with antibodys to p57Kip2 or Skp2. (B) Cells were transfected with HA-Skp2 and Flag-p57 (WT) or Flag-p57 (T310A) for 48 hours and then incubated with 10 μM MG132 for 4 hours. After that, cell extracts were immunoprecipitated with an antibody against Flag and analyzed by immunoblotting. (C) After transfection with HA-skp2 and Flag-p57 (WT) or Flag-p57 (T310A) for 48 hours, cells were incubated for the indicated times with CHX (20 μg/mL). Cell lysates were then used for immunoblot analysis with antibodies to Flag or HA. β-Actin was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4525793&req=5

f4-bcbcr-suppl.1-2015-013: Association of Skp2 with p57Kip2 degradation in breast cancer cell lines. (A) After transfection with Skp2 siRNA or control for 48 hours, T47D cells were incubated for the indicated times with CHX (20 μg/mL). Cell lysates were then subjected to immunoblot analysis with antibodys to p57Kip2 or Skp2. (B) Cells were transfected with HA-Skp2 and Flag-p57 (WT) or Flag-p57 (T310A) for 48 hours and then incubated with 10 μM MG132 for 4 hours. After that, cell extracts were immunoprecipitated with an antibody against Flag and analyzed by immunoblotting. (C) After transfection with HA-skp2 and Flag-p57 (WT) or Flag-p57 (T310A) for 48 hours, cells were incubated for the indicated times with CHX (20 μg/mL). Cell lysates were then used for immunoblot analysis with antibodies to Flag or HA. β-Actin was used as a loading control.
Mentions: We hypothesized that Skp2 regulates the stability of p57Kip2 in breast cancer. T47D breast cancer cells were transfected with siRNA-Skp2 or control, and then incubated with the protein synthesis inhibitor CHX (cycloheximide, 20 μg/mL) for 0, 1, 2, and 4 hours. After exposure to CHX, T47D cells exhibited an almost complete loss of p57Kip2 within two hours. The degradation of endogenous p57Kip2 was much slower in Skp2-knockdown T47D cells compared with the control cells (Fig. 4A). These results indicate that knockdown of Skp2 inhibits the degradation of p57Kip2 in T47D cells.

Bottom Line: Downregulation of p57(Kip2) is involved in tumor progression, and S-phase kinase-associated protein 2 (Skp2) is an E3 ligase that regulates a variety of cell cycle proteins.In addition, univariate analysis showed that Skp2, p57(Kip2), histological grade, lymph node metastasis, and estrogen and progesterone receptors (ER and PR) were all associated with DFS, and multivariate analysis revealed that lymph node metastasis and Skp2 were independent prognostic biomarkers.Half-life and immunoprecipitation (IP) experiments indicated that Skp2 directly interacts with p57(Kip2) and promotes its degradation, rather than its mutant p57(Kip2) (T310A).

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, First Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an, Shannxi, P. R. China.

ABSTRACT
Downregulation of p57(Kip2) is involved in tumor progression, and S-phase kinase-associated protein 2 (Skp2) is an E3 ligase that regulates a variety of cell cycle proteins. However, the prognostic value of p57(Kip2) and its correlation with Skp2 in breast cancer have not been fully elucidated. Here we report our study on the expression of p57(Kip2) and Skp2 in 102 breast cancer patients by immunohistochemistry, and analysis of clinicopathologic parameters in relation to patient prognosis. The expression of p57(Kip2) was negatively associated with Skp2 expression in breast cancer (r = -0.26, P = 0.009). Kaplan-Meier analysis indicated that both high Skp2 and low p57(Kip2) correlated with poor disease-free survival (DFS) (P = 0.05), and a group with the combination of high Skp2/low p57(Kip2) demonstrated even worse DFS (log-rank = 21.118, P < 0.001). In addition, univariate analysis showed that Skp2, p57(Kip2), histological grade, lymph node metastasis, and estrogen and progesterone receptors (ER and PR) were all associated with DFS, and multivariate analysis revealed that lymph node metastasis and Skp2 were independent prognostic biomarkers. The correlation between p57 and Skp2 was further demonstrated in multiple breast cancer cell lines and cell cycle phases. Half-life and immunoprecipitation (IP) experiments indicated that Skp2 directly interacts with p57(Kip2) and promotes its degradation, rather than its mutant p57(Kip2) (T310A). Overall, our findings demonstrate that Skp2 directly degrades p57(Kip2), and an inverse correlation between these proteins (high skp2/low p57(Kip2)) is associated with poor prognosis in breast cancer. Thus, our results indicate a combined prognostic value of these markers in breast cancer diagnosis and treatment.

No MeSH data available.


Related in: MedlinePlus