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IκB kinases increase Myc protein stability and enhance progression of breast cancer cells.

Yeh PY, Lu YS, Ou DL, Cheng AL - Mol. Cancer (2011)

Bottom Line: Consequently, these treatments decrease the tumorigenic and invasive ability of breast cancer cells.Inhibition of IKKs prevents these doxorubicin-induced effects.Our study indicates that IKKs tightly regulate Myc expression through prolonging protein stability, and suggests that IKKs are potentially therapeutic targets and that suppression of IKKs may be used following chemotherapy to reduce the risk of treatment-induced tumor progression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oncology, National Taiwan University Hospital, No, 7, Chung-Shan South Road, Taipei, 100, Taiwan.

ABSTRACT

Background: Both IκB kinase (IKK) complex and oncgenic protein Myc play important roles in cancer progression, including cancer cell invasiveness and metastasis. The levels of Myc is regulated by the phosphorylation of Myc at Thr58 and Ser62.

Results: In this study, we show that the expression of Myc is associated with IKKα and IKKβ in breast cancers and that Myc is an IKKs substrate. Suppression of IKK activity by either chemical inhibitor or transfection of kinase-dead mutants decreases the phosphorylation of Myc at Ser62 and enhances the degradation of Myc. Consequently, these treatments decrease the tumorigenic and invasive ability of breast cancer cells. Furthermore, doxorubicin, a frequently used anticancer drug in breast cancer, activates IKKs and Myc, thereby increasing invasiveness and tumorigenesis of breast carcinoma MCF7 cells. Inhibition of IKKs prevents these doxorubicin-induced effects.

Conclusions: Our study indicates that IKKs tightly regulate Myc expression through prolonging protein stability, and suggests that IKKs are potentially therapeutic targets and that suppression of IKKs may be used following chemotherapy to reduce the risk of treatment-induced tumor progression.

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IKKα and IKKβ increase tumorigenesis and invasiveness of MCF7 cells. (A) MTT assay was used to analyze the growth rate of indicated cells. Each data represents mean ± SD calculated from three independent experiments, and there are four wells for each point in a single experiment. (B) Assay of colony-forming ability in soft-agar. Each experiment have three 60 mm dishes and each data represents mean ± SD calculated from two independent experiments. (C) Assay of invasive ability of indicated cells. Each experiment have three trans-wells and each data represents mean ± SD calculated from two independent experiments. (D) Western blot analysis of the expression of cyclin D1 and twist protein in the indicated cells.
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Figure 5: IKKα and IKKβ increase tumorigenesis and invasiveness of MCF7 cells. (A) MTT assay was used to analyze the growth rate of indicated cells. Each data represents mean ± SD calculated from three independent experiments, and there are four wells for each point in a single experiment. (B) Assay of colony-forming ability in soft-agar. Each experiment have three 60 mm dishes and each data represents mean ± SD calculated from two independent experiments. (C) Assay of invasive ability of indicated cells. Each experiment have three trans-wells and each data represents mean ± SD calculated from two independent experiments. (D) Western blot analysis of the expression of cyclin D1 and twist protein in the indicated cells.

Mentions: We next characterized the biological functions of IKKs by analyzing IKK-transfected cells. Cells overexpressing either wild-type or kinase-dead IKKα or IKKβ showed comparable growth rates (Figure 5A). However, wild-type IKKα and IKKβ increased cell growth in soft-agar, a well-defined character of tumorigenesis, and kinase-dead IKKα and IKKβ suppressed this ability of the cells (Figure 5B). Next, we assayed the ability of IKK-overexpressing cells to pass through matrigel, a well-established method to determine the invasive activity of cancer cells. Wild-type IKKα or IKKβ overexpressing cells showed a higher invasive ability than parental MCF7 cells; by contrast, the invasive ability was decreased in kinase-dead IKKα or IKKβ-transfected cells (Figure 5C). Because cyclin D1 and Twist are two important down-stream effectors of Myc and their biological functions are associated with the invasive/tumorigenic ability of cancer cells [20,33,34], cyclin D1 and Twist protein levels were determined by Western blot analysis. Consistently, the levels of these two proteins were increased in wild-type IKKα- or IKKβ- transfected cells and decreased in kinase-dead IKKα-or IKKβ- transfected cells (Figure 5D).


IκB kinases increase Myc protein stability and enhance progression of breast cancer cells.

Yeh PY, Lu YS, Ou DL, Cheng AL - Mol. Cancer (2011)

IKKα and IKKβ increase tumorigenesis and invasiveness of MCF7 cells. (A) MTT assay was used to analyze the growth rate of indicated cells. Each data represents mean ± SD calculated from three independent experiments, and there are four wells for each point in a single experiment. (B) Assay of colony-forming ability in soft-agar. Each experiment have three 60 mm dishes and each data represents mean ± SD calculated from two independent experiments. (C) Assay of invasive ability of indicated cells. Each experiment have three trans-wells and each data represents mean ± SD calculated from two independent experiments. (D) Western blot analysis of the expression of cyclin D1 and twist protein in the indicated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3104363&req=5

Figure 5: IKKα and IKKβ increase tumorigenesis and invasiveness of MCF7 cells. (A) MTT assay was used to analyze the growth rate of indicated cells. Each data represents mean ± SD calculated from three independent experiments, and there are four wells for each point in a single experiment. (B) Assay of colony-forming ability in soft-agar. Each experiment have three 60 mm dishes and each data represents mean ± SD calculated from two independent experiments. (C) Assay of invasive ability of indicated cells. Each experiment have three trans-wells and each data represents mean ± SD calculated from two independent experiments. (D) Western blot analysis of the expression of cyclin D1 and twist protein in the indicated cells.
Mentions: We next characterized the biological functions of IKKs by analyzing IKK-transfected cells. Cells overexpressing either wild-type or kinase-dead IKKα or IKKβ showed comparable growth rates (Figure 5A). However, wild-type IKKα and IKKβ increased cell growth in soft-agar, a well-defined character of tumorigenesis, and kinase-dead IKKα and IKKβ suppressed this ability of the cells (Figure 5B). Next, we assayed the ability of IKK-overexpressing cells to pass through matrigel, a well-established method to determine the invasive activity of cancer cells. Wild-type IKKα or IKKβ overexpressing cells showed a higher invasive ability than parental MCF7 cells; by contrast, the invasive ability was decreased in kinase-dead IKKα or IKKβ-transfected cells (Figure 5C). Because cyclin D1 and Twist are two important down-stream effectors of Myc and their biological functions are associated with the invasive/tumorigenic ability of cancer cells [20,33,34], cyclin D1 and Twist protein levels were determined by Western blot analysis. Consistently, the levels of these two proteins were increased in wild-type IKKα- or IKKβ- transfected cells and decreased in kinase-dead IKKα-or IKKβ- transfected cells (Figure 5D).

Bottom Line: Consequently, these treatments decrease the tumorigenic and invasive ability of breast cancer cells.Inhibition of IKKs prevents these doxorubicin-induced effects.Our study indicates that IKKs tightly regulate Myc expression through prolonging protein stability, and suggests that IKKs are potentially therapeutic targets and that suppression of IKKs may be used following chemotherapy to reduce the risk of treatment-induced tumor progression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oncology, National Taiwan University Hospital, No, 7, Chung-Shan South Road, Taipei, 100, Taiwan.

ABSTRACT

Background: Both IκB kinase (IKK) complex and oncgenic protein Myc play important roles in cancer progression, including cancer cell invasiveness and metastasis. The levels of Myc is regulated by the phosphorylation of Myc at Thr58 and Ser62.

Results: In this study, we show that the expression of Myc is associated with IKKα and IKKβ in breast cancers and that Myc is an IKKs substrate. Suppression of IKK activity by either chemical inhibitor or transfection of kinase-dead mutants decreases the phosphorylation of Myc at Ser62 and enhances the degradation of Myc. Consequently, these treatments decrease the tumorigenic and invasive ability of breast cancer cells. Furthermore, doxorubicin, a frequently used anticancer drug in breast cancer, activates IKKs and Myc, thereby increasing invasiveness and tumorigenesis of breast carcinoma MCF7 cells. Inhibition of IKKs prevents these doxorubicin-induced effects.

Conclusions: Our study indicates that IKKs tightly regulate Myc expression through prolonging protein stability, and suggests that IKKs are potentially therapeutic targets and that suppression of IKKs may be used following chemotherapy to reduce the risk of treatment-induced tumor progression.

Show MeSH
Related in: MedlinePlus