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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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Related in: MedlinePlus

IKKs-Myc pathway is inducible. (A) MCF7 cells were treated with 10 μM Bay11-0782 alone, or 200 nM doxorubicin in the presence or absence of 10 μM Bay11-0782 for 24 hours (Dox, Bay, and Bay/Dox), or 3-hr short-term pulse followed by 24 hours release in the presence or absence of Bay11-0782 (Dox3-R, Bay/Dox3-R, and Bay/Dox3-R/Bay). Whole cells lysates were subjected to Western blot analysis. (B) MCF7 cells were treated 200 nM doxorubicin with or without 10 μM Bay11-0782 for 24 hours. The cytoplasmic and nuclear lysates were subjected to Western blotting. (C) MCF7 cells were treated with 200 nM doxorubicin in the presence or absence of 10 μM Bay11-0782 for 3 hours. The cells were then seeded into soft-agar in complete medium with or without 10 μM Bay11-0782 for two weeks. Each experiment have three 60 mm dishes and each data represents mean ± SD calculated from two independent experiments. (D) MCF7 cells were treated as shown, and then the ability of cells to penetrate matrigel was determined. Each experiment have three trans-wells and each data represents mean ± SD calculated from two independent experiments.
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Figure 6: IKKs-Myc pathway is inducible. (A) MCF7 cells were treated with 10 μM Bay11-0782 alone, or 200 nM doxorubicin in the presence or absence of 10 μM Bay11-0782 for 24 hours (Dox, Bay, and Bay/Dox), or 3-hr short-term pulse followed by 24 hours release in the presence or absence of Bay11-0782 (Dox3-R, Bay/Dox3-R, and Bay/Dox3-R/Bay). Whole cells lysates were subjected to Western blot analysis. (B) MCF7 cells were treated 200 nM doxorubicin with or without 10 μM Bay11-0782 for 24 hours. The cytoplasmic and nuclear lysates were subjected to Western blotting. (C) MCF7 cells were treated with 200 nM doxorubicin in the presence or absence of 10 μM Bay11-0782 for 3 hours. The cells were then seeded into soft-agar in complete medium with or without 10 μM Bay11-0782 for two weeks. Each experiment have three 60 mm dishes and each data represents mean ± SD calculated from two independent experiments. (D) MCF7 cells were treated as shown, and then the ability of cells to penetrate matrigel was determined. Each experiment have three trans-wells and each data represents mean ± SD calculated from two independent experiments.

Mentions: Because IKKs play an important role in tumor cells response to various stresses, it was of interest to ask whether common chemotherapeutic agents for breast cancer treatment could induce IKK and Myc activation. We used doxorubicin (200 nM; IC50 for MCF7 cells) alone or combined with Bay11-0782 to treat MCF7 cells. Cells were exposed to doxorubicin with or without Bay11-0782 for either 24-hour or for 3-hour followed by a 24-hour release in the presence or absence of Bay11-0782. Doxorubicin induced IKKs activation, with induction levels highest in cells released from a 3-houre doxorubicin treatment. When Bay11-0782 was re-added to the cells released from a 3-hour treatment with doxorubicin with or without Bay11-0782, decreased levels of doxorubicin-induced IKKs activation were observed. IKK activity returned to control levels after a 24-hour Bay11-0782 treatment. (Figure 6A). Consequently, the phosphorylation of Myc at Ser 62 was increased by doxorubicin, and the highest level of induction was achieved in cells released from a 3-hour doxorubicin treatment with or without Bay11-0782 cotreatment. Re-addition of Bay11-0782 blocked this induction. The phosphorylation of Myc at Thr58 remained at a relatively constant level following all treatments. The level of Myc protein was changed with a consistent pattern as the status of phosphorylated Myc Ser62. Cyclin D1 and Twist levels were also altered in a similar manner (Figure 6A). In addition, Western blot analysis showed that NF-κB was not affected by these treatments (Figure 6B).


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

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

IKKs-Myc pathway is inducible. (A) MCF7 cells were treated with 10 μM Bay11-0782 alone, or 200 nM doxorubicin in the presence or absence of 10 μM Bay11-0782 for 24 hours (Dox, Bay, and Bay/Dox), or 3-hr short-term pulse followed by 24 hours release in the presence or absence of Bay11-0782 (Dox3-R, Bay/Dox3-R, and Bay/Dox3-R/Bay). Whole cells lysates were subjected to Western blot analysis. (B) MCF7 cells were treated 200 nM doxorubicin with or without 10 μM Bay11-0782 for 24 hours. The cytoplasmic and nuclear lysates were subjected to Western blotting. (C) MCF7 cells were treated with 200 nM doxorubicin in the presence or absence of 10 μM Bay11-0782 for 3 hours. The cells were then seeded into soft-agar in complete medium with or without 10 μM Bay11-0782 for two weeks. Each experiment have three 60 mm dishes and each data represents mean ± SD calculated from two independent experiments. (D) MCF7 cells were treated as shown, and then the ability of cells to penetrate matrigel was determined. Each experiment have three trans-wells and each data represents mean ± SD calculated from two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 6: IKKs-Myc pathway is inducible. (A) MCF7 cells were treated with 10 μM Bay11-0782 alone, or 200 nM doxorubicin in the presence or absence of 10 μM Bay11-0782 for 24 hours (Dox, Bay, and Bay/Dox), or 3-hr short-term pulse followed by 24 hours release in the presence or absence of Bay11-0782 (Dox3-R, Bay/Dox3-R, and Bay/Dox3-R/Bay). Whole cells lysates were subjected to Western blot analysis. (B) MCF7 cells were treated 200 nM doxorubicin with or without 10 μM Bay11-0782 for 24 hours. The cytoplasmic and nuclear lysates were subjected to Western blotting. (C) MCF7 cells were treated with 200 nM doxorubicin in the presence or absence of 10 μM Bay11-0782 for 3 hours. The cells were then seeded into soft-agar in complete medium with or without 10 μM Bay11-0782 for two weeks. Each experiment have three 60 mm dishes and each data represents mean ± SD calculated from two independent experiments. (D) MCF7 cells were treated as shown, and then the ability of cells to penetrate matrigel was determined. Each experiment have three trans-wells and each data represents mean ± SD calculated from two independent experiments.
Mentions: Because IKKs play an important role in tumor cells response to various stresses, it was of interest to ask whether common chemotherapeutic agents for breast cancer treatment could induce IKK and Myc activation. We used doxorubicin (200 nM; IC50 for MCF7 cells) alone or combined with Bay11-0782 to treat MCF7 cells. Cells were exposed to doxorubicin with or without Bay11-0782 for either 24-hour or for 3-hour followed by a 24-hour release in the presence or absence of Bay11-0782. Doxorubicin induced IKKs activation, with induction levels highest in cells released from a 3-houre doxorubicin treatment. When Bay11-0782 was re-added to the cells released from a 3-hour treatment with doxorubicin with or without Bay11-0782, decreased levels of doxorubicin-induced IKKs activation were observed. IKK activity returned to control levels after a 24-hour Bay11-0782 treatment. (Figure 6A). Consequently, the phosphorylation of Myc at Ser 62 was increased by doxorubicin, and the highest level of induction was achieved in cells released from a 3-hour doxorubicin treatment with or without Bay11-0782 cotreatment. Re-addition of Bay11-0782 blocked this induction. The phosphorylation of Myc at Thr58 remained at a relatively constant level following all treatments. The level of Myc protein was changed with a consistent pattern as the status of phosphorylated Myc Ser62. Cyclin D1 and Twist levels were also altered in a similar manner (Figure 6A). In addition, Western blot analysis showed that NF-κB was not affected by these treatments (Figure 6B).

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