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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 Myc protein stability. The stability of Myc protein was analyzed by Western blot analysis of the whole cell lysates prepared from (A) MCF7 with or without a 12 hours, 10 μM Bay11-0782 treatment (B) wild-type and kinase-dead IKKα transfected cells (C) wild-type and kinase-dead IKKβ transfected cells along a time course after adding cycloheximide. The stain of tubulin was used as loading control. The reading of Myc density was normalized to the reading of tubulin density. Each data represents mean ± SD calculated from two independent experiments. Whole cell lysates were prepared from (D) MCF7 cells, (E) wild-type and kinase-dead IKKα transfected cells, and (F) wild-type and kinase-dead IKKβ transfected cells were subjected to coimmuoprecipitation using indicated antibodies. The precipitated complex was further Western blot analyzed the corresponding proteins shown in the figure. (G) Confocal microscopy observation. Breast cancer tissues which were previously identified positive or negative for IKKα, IKKβ and Myc expressions by IHC staining were used. The slides were dual stained with rabbit anti-IKKα/mouse anti-Myc antibodies coupled with FITC-conjugated goat anti-rabbit IgG/Rodamine-conjugated donkey anti-mouse IgG antibodies or goat anti-IKKβ/mouse anti-Myc antibodies coupled with FITC-conjugated donkey anti-goat IgG/Rodamine-conjugated donkey anti-mouse IgG antibodies, respectively.
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Figure 4: IKKα and IKKβ increase Myc protein stability. The stability of Myc protein was analyzed by Western blot analysis of the whole cell lysates prepared from (A) MCF7 with or without a 12 hours, 10 μM Bay11-0782 treatment (B) wild-type and kinase-dead IKKα transfected cells (C) wild-type and kinase-dead IKKβ transfected cells along a time course after adding cycloheximide. The stain of tubulin was used as loading control. The reading of Myc density was normalized to the reading of tubulin density. Each data represents mean ± SD calculated from two independent experiments. Whole cell lysates were prepared from (D) MCF7 cells, (E) wild-type and kinase-dead IKKα transfected cells, and (F) wild-type and kinase-dead IKKβ transfected cells were subjected to coimmuoprecipitation using indicated antibodies. The precipitated complex was further Western blot analyzed the corresponding proteins shown in the figure. (G) Confocal microscopy observation. Breast cancer tissues which were previously identified positive or negative for IKKα, IKKβ and Myc expressions by IHC staining were used. The slides were dual stained with rabbit anti-IKKα/mouse anti-Myc antibodies coupled with FITC-conjugated goat anti-rabbit IgG/Rodamine-conjugated donkey anti-mouse IgG antibodies or goat anti-IKKβ/mouse anti-Myc antibodies coupled with FITC-conjugated donkey anti-goat IgG/Rodamine-conjugated donkey anti-mouse IgG antibodies, respectively.

Mentions: Next, we determined the degradation rate of the Myc protein in Bay11-0782 treated MCF7 cells and in IKK- transfected cells by Western blot analysis along a time course after adding a protein synthesis inhibitor, cycloheximide. Bay11-7082 induced a more rapid degradation rate of Myc protein (Figure 4A). Wild-type IKKα or IKKβ increased the stability of Myc protein. On the other hand, kinase-dead IKKα or IKKβ enhanced Myc protein degradation (Figure 4B and 4C).


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 Myc protein stability. The stability of Myc protein was analyzed by Western blot analysis of the whole cell lysates prepared from (A) MCF7 with or without a 12 hours, 10 μM Bay11-0782 treatment (B) wild-type and kinase-dead IKKα transfected cells (C) wild-type and kinase-dead IKKβ transfected cells along a time course after adding cycloheximide. The stain of tubulin was used as loading control. The reading of Myc density was normalized to the reading of tubulin density. Each data represents mean ± SD calculated from two independent experiments. Whole cell lysates were prepared from (D) MCF7 cells, (E) wild-type and kinase-dead IKKα transfected cells, and (F) wild-type and kinase-dead IKKβ transfected cells were subjected to coimmuoprecipitation using indicated antibodies. The precipitated complex was further Western blot analyzed the corresponding proteins shown in the figure. (G) Confocal microscopy observation. Breast cancer tissues which were previously identified positive or negative for IKKα, IKKβ and Myc expressions by IHC staining were used. The slides were dual stained with rabbit anti-IKKα/mouse anti-Myc antibodies coupled with FITC-conjugated goat anti-rabbit IgG/Rodamine-conjugated donkey anti-mouse IgG antibodies or goat anti-IKKβ/mouse anti-Myc antibodies coupled with FITC-conjugated donkey anti-goat IgG/Rodamine-conjugated donkey anti-mouse IgG antibodies, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: IKKα and IKKβ increase Myc protein stability. The stability of Myc protein was analyzed by Western blot analysis of the whole cell lysates prepared from (A) MCF7 with or without a 12 hours, 10 μM Bay11-0782 treatment (B) wild-type and kinase-dead IKKα transfected cells (C) wild-type and kinase-dead IKKβ transfected cells along a time course after adding cycloheximide. The stain of tubulin was used as loading control. The reading of Myc density was normalized to the reading of tubulin density. Each data represents mean ± SD calculated from two independent experiments. Whole cell lysates were prepared from (D) MCF7 cells, (E) wild-type and kinase-dead IKKα transfected cells, and (F) wild-type and kinase-dead IKKβ transfected cells were subjected to coimmuoprecipitation using indicated antibodies. The precipitated complex was further Western blot analyzed the corresponding proteins shown in the figure. (G) Confocal microscopy observation. Breast cancer tissues which were previously identified positive or negative for IKKα, IKKβ and Myc expressions by IHC staining were used. The slides were dual stained with rabbit anti-IKKα/mouse anti-Myc antibodies coupled with FITC-conjugated goat anti-rabbit IgG/Rodamine-conjugated donkey anti-mouse IgG antibodies or goat anti-IKKβ/mouse anti-Myc antibodies coupled with FITC-conjugated donkey anti-goat IgG/Rodamine-conjugated donkey anti-mouse IgG antibodies, respectively.
Mentions: Next, we determined the degradation rate of the Myc protein in Bay11-0782 treated MCF7 cells and in IKK- transfected cells by Western blot analysis along a time course after adding a protein synthesis inhibitor, cycloheximide. Bay11-7082 induced a more rapid degradation rate of Myc protein (Figure 4A). Wild-type IKKα or IKKβ increased the stability of Myc protein. On the other hand, kinase-dead IKKα or IKKβ enhanced Myc protein degradation (Figure 4B and 4C).

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