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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 in a transcription-independent manner. (A) MCF7 cells were treated with 10 μM Bay11-0782 for 12 hours. The RNA was extracted from indicated cells and random-primed reverse transcribed into cDNA. The relative expression of Myc was determined by qPCR. The expression of GAPDH was used as an internal control. The specificity of the primer set for Myc and GAPDH was demonstrated by agarose electrophoresis of PCR product (inserted figure) and by analysis of dissociation curve (data not shown). Each data represents mean ± SD calculated from two independent experiments. To determine the degradation rate of Myc mRNA, the cDNAs were prepared from (B) MCF7 cells were treated with 10 μM Bay11-0782 for 12 hours, (C) MCF7, wild-type IKKα and IKKβ transfected cells, and (D) MCF7, kinase-dead IKKα and IKKβ transfected cells. The relative level of Myc mRNA was determined by qPCR and expressed along a time course after adding actinimycin D. Each data represents mean ± SD calculated from two independent experiments.
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Figure 3: IKKα and IKKβ increase Myc in a transcription-independent manner. (A) MCF7 cells were treated with 10 μM Bay11-0782 for 12 hours. The RNA was extracted from indicated cells and random-primed reverse transcribed into cDNA. The relative expression of Myc was determined by qPCR. The expression of GAPDH was used as an internal control. The specificity of the primer set for Myc and GAPDH was demonstrated by agarose electrophoresis of PCR product (inserted figure) and by analysis of dissociation curve (data not shown). Each data represents mean ± SD calculated from two independent experiments. To determine the degradation rate of Myc mRNA, the cDNAs were prepared from (B) MCF7 cells were treated with 10 μM Bay11-0782 for 12 hours, (C) MCF7, wild-type IKKα and IKKβ transfected cells, and (D) MCF7, kinase-dead IKKα and IKKβ transfected cells. The relative level of Myc mRNA was determined by qPCR and expressed along a time course after adding actinimycin D. Each data represents mean ± SD calculated from two independent experiments.

Mentions: To identify whether IKKα or IKKβ could increase the transcription of Myc mRNA, qPCR was used to determine the relative levels of Myc mRNA in Bay11-7082 treated MCF7 cells and IKK-transfected cells (Figure 3A). Overexpression of either wild-type IKKα or IKKβ slightly increased the level of Myc mRNA. The induction level was less than two-fold of the control which is the cut-point of most gene array analyses. Interestingly, while Bay11-7082 only marginally reduced Myc mRNA levels, a small increase of Myc mRNA (less than 1.5-fold of control levels) was observed in both kinase-dead IKKα- and IKKβ-transfected cells. These results suggested that IKKs could increase Myc protein levels in a transcription-independent manner.


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 in a transcription-independent manner. (A) MCF7 cells were treated with 10 μM Bay11-0782 for 12 hours. The RNA was extracted from indicated cells and random-primed reverse transcribed into cDNA. The relative expression of Myc was determined by qPCR. The expression of GAPDH was used as an internal control. The specificity of the primer set for Myc and GAPDH was demonstrated by agarose electrophoresis of PCR product (inserted figure) and by analysis of dissociation curve (data not shown). Each data represents mean ± SD calculated from two independent experiments. To determine the degradation rate of Myc mRNA, the cDNAs were prepared from (B) MCF7 cells were treated with 10 μM Bay11-0782 for 12 hours, (C) MCF7, wild-type IKKα and IKKβ transfected cells, and (D) MCF7, kinase-dead IKKα and IKKβ transfected cells. The relative level of Myc mRNA was determined by qPCR and expressed along a time course after adding actinimycin D. Each data represents mean ± SD calculated from two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: IKKα and IKKβ increase Myc in a transcription-independent manner. (A) MCF7 cells were treated with 10 μM Bay11-0782 for 12 hours. The RNA was extracted from indicated cells and random-primed reverse transcribed into cDNA. The relative expression of Myc was determined by qPCR. The expression of GAPDH was used as an internal control. The specificity of the primer set for Myc and GAPDH was demonstrated by agarose electrophoresis of PCR product (inserted figure) and by analysis of dissociation curve (data not shown). Each data represents mean ± SD calculated from two independent experiments. To determine the degradation rate of Myc mRNA, the cDNAs were prepared from (B) MCF7 cells were treated with 10 μM Bay11-0782 for 12 hours, (C) MCF7, wild-type IKKα and IKKβ transfected cells, and (D) MCF7, kinase-dead IKKα and IKKβ transfected cells. The relative level of Myc mRNA was determined by qPCR and expressed along a time course after adding actinimycin D. Each data represents mean ± SD calculated from two independent experiments.
Mentions: To identify whether IKKα or IKKβ could increase the transcription of Myc mRNA, qPCR was used to determine the relative levels of Myc mRNA in Bay11-7082 treated MCF7 cells and IKK-transfected cells (Figure 3A). Overexpression of either wild-type IKKα or IKKβ slightly increased the level of Myc mRNA. The induction level was less than two-fold of the control which is the cut-point of most gene array analyses. Interestingly, while Bay11-7082 only marginally reduced Myc mRNA levels, a small increase of Myc mRNA (less than 1.5-fold of control levels) was observed in both kinase-dead IKKα- and IKKβ-transfected cells. These results suggested that IKKs could increase Myc protein levels in a transcription-independent manner.

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