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Expression of HER-2 in MCF-7 breast cancer cells modulates anti-apoptotic proteins Survivin and Bcl-2 via the extracellular signal-related kinase (ERK) and phosphoinositide-3 kinase (PI3K) signalling pathways.

Siddiqa A, Long LM, Li L, Marciniak RA, Kazhdan I - BMC Cancer (2008)

Bottom Line: We did not observe a change in survivin and NF-kappaB promoter activity in HER-2 expressing MCF7 cells.Our results indicate that short term over expression of HER-2 up regulates antiapoptotic proteins Bcl-2 and survivin in MCF7 cells.Additionally we show that survivin up-regulation is not at transcriptional level.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, University of Texas Health Science Center, San Antonio, TX 78229, USA. siddiqa@uthscsa.edu

ABSTRACT

Background: The oncoprotein HER-2 is over-expressed and/or has undergone gene amplification in between 20 to 30% of breast and ovarian cancers. HER-2 amplified breast cancer is associated with a poor prognosis and increased resistance to chemo- and hormonal therapy. Data supporting the transforming potential of HER-2 are irrefutable but the mechanism by which HER-2 contributes to this process is complex and a unified model of HER2-induced increased cell proliferation and survival has not emerged.To understand the initial event(s) that take place by HER-2 over expression, we studied the effect of short term induction of HER-2 expression in the MCF7 breast cancer cell line.

Methods: We examined the modulation of apoptotic pathways by tetracycline-regulated HER-2 expression for 48 hrs in the MCF7 breast cancer cell line. Specific inhibitors were used to determine signalling pathways that are required for HER-2 induced up-regulation of survivin.

Results: Tetracycline regulated short term over expression of HER-2 in the MCF7 cell line increased the antiapoptotic proteins Bcl-2 and survivin levels. Significant increase of extracellular signal-related kinase (ERK) activation but not AKT1, AKT2 and STAT3 was observed in HER-2 over-expressing MCF7 cells. Specific inhibitors of ERK, and phosphoinositide-3 kinase (PI3K), inhibited the HER-2 induced up-regulation of survivin. We did not observe a change in survivin and NF-kappaB promoter activity in HER-2 expressing MCF7 cells.

Conclusion: Our results indicate that short term over expression of HER-2 up regulates antiapoptotic proteins Bcl-2 and survivin in MCF7 cells. We determined that survivin is up-regulated via ERK activation and PI3K signalling. Additionally we show that survivin up-regulation is not at transcriptional level. These data provide insight into the mechanism(s) by which induction of HER-2 over expression up-regulates survivin and Bcl-2 and identifies new targets for therapy of breast cancer.

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Related in: MedlinePlus

Analysis of total ERK 1/2, STAT 3, AKT 1 and their phosphorylated forms in HER2-overexpressing and control cells. pTRE-HER2 and pTRE-transfected MCF-7 clones were incubated with or without doxycycline for 48 hours. Whole cell extracts were prepared and analyzed by Western blotting with the indicated antibodies. Experiments were repeated twice with independently collected extracts.
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Figure 5: Analysis of total ERK 1/2, STAT 3, AKT 1 and their phosphorylated forms in HER2-overexpressing and control cells. pTRE-HER2 and pTRE-transfected MCF-7 clones were incubated with or without doxycycline for 48 hours. Whole cell extracts were prepared and analyzed by Western blotting with the indicated antibodies. Experiments were repeated twice with independently collected extracts.

Mentions: Over-expression of HER-2 has been reported to activate multiple signal transduction pathways including Ras-MAPK, the PI3-K-Akt-NF-kappaB cascade and STAT 3 [24]. We determined the state of activation of these pathways in MCF7 cells after doxycycline-induced HER-2 overexpression. Vector-transfected (pTRE) cells and MCF7-HER2 cells were incubated with or without doxycycline for 48 hours. Whole cell protein extracts were analyzed by western blotting for expression of total ERK 1/2, STAT 3 and AKT 1 and their respective activated forms (phospho-ERK 1/2, phospho-STAT3 and phospho-AKT-1; Figure 5). There were no changes in the total levels of ERK 1/2, STAT 3 and AKT 1. In accordance with the previous reports, we identified pronounced induction of phospho-ERK 1/2 in doxycycline-treated MCF7-HER2 cells. However, there were no changes in the levels of phospho-STAT 3 and phospho-AKT (Ser473).


Expression of HER-2 in MCF-7 breast cancer cells modulates anti-apoptotic proteins Survivin and Bcl-2 via the extracellular signal-related kinase (ERK) and phosphoinositide-3 kinase (PI3K) signalling pathways.

Siddiqa A, Long LM, Li L, Marciniak RA, Kazhdan I - BMC Cancer (2008)

Analysis of total ERK 1/2, STAT 3, AKT 1 and their phosphorylated forms in HER2-overexpressing and control cells. pTRE-HER2 and pTRE-transfected MCF-7 clones were incubated with or without doxycycline for 48 hours. Whole cell extracts were prepared and analyzed by Western blotting with the indicated antibodies. Experiments were repeated twice with independently collected extracts.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2386479&req=5

Figure 5: Analysis of total ERK 1/2, STAT 3, AKT 1 and their phosphorylated forms in HER2-overexpressing and control cells. pTRE-HER2 and pTRE-transfected MCF-7 clones were incubated with or without doxycycline for 48 hours. Whole cell extracts were prepared and analyzed by Western blotting with the indicated antibodies. Experiments were repeated twice with independently collected extracts.
Mentions: Over-expression of HER-2 has been reported to activate multiple signal transduction pathways including Ras-MAPK, the PI3-K-Akt-NF-kappaB cascade and STAT 3 [24]. We determined the state of activation of these pathways in MCF7 cells after doxycycline-induced HER-2 overexpression. Vector-transfected (pTRE) cells and MCF7-HER2 cells were incubated with or without doxycycline for 48 hours. Whole cell protein extracts were analyzed by western blotting for expression of total ERK 1/2, STAT 3 and AKT 1 and their respective activated forms (phospho-ERK 1/2, phospho-STAT3 and phospho-AKT-1; Figure 5). There were no changes in the total levels of ERK 1/2, STAT 3 and AKT 1. In accordance with the previous reports, we identified pronounced induction of phospho-ERK 1/2 in doxycycline-treated MCF7-HER2 cells. However, there were no changes in the levels of phospho-STAT 3 and phospho-AKT (Ser473).

Bottom Line: We did not observe a change in survivin and NF-kappaB promoter activity in HER-2 expressing MCF7 cells.Our results indicate that short term over expression of HER-2 up regulates antiapoptotic proteins Bcl-2 and survivin in MCF7 cells.Additionally we show that survivin up-regulation is not at transcriptional level.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, University of Texas Health Science Center, San Antonio, TX 78229, USA. siddiqa@uthscsa.edu

ABSTRACT

Background: The oncoprotein HER-2 is over-expressed and/or has undergone gene amplification in between 20 to 30% of breast and ovarian cancers. HER-2 amplified breast cancer is associated with a poor prognosis and increased resistance to chemo- and hormonal therapy. Data supporting the transforming potential of HER-2 are irrefutable but the mechanism by which HER-2 contributes to this process is complex and a unified model of HER2-induced increased cell proliferation and survival has not emerged.To understand the initial event(s) that take place by HER-2 over expression, we studied the effect of short term induction of HER-2 expression in the MCF7 breast cancer cell line.

Methods: We examined the modulation of apoptotic pathways by tetracycline-regulated HER-2 expression for 48 hrs in the MCF7 breast cancer cell line. Specific inhibitors were used to determine signalling pathways that are required for HER-2 induced up-regulation of survivin.

Results: Tetracycline regulated short term over expression of HER-2 in the MCF7 cell line increased the antiapoptotic proteins Bcl-2 and survivin levels. Significant increase of extracellular signal-related kinase (ERK) activation but not AKT1, AKT2 and STAT3 was observed in HER-2 over-expressing MCF7 cells. Specific inhibitors of ERK, and phosphoinositide-3 kinase (PI3K), inhibited the HER-2 induced up-regulation of survivin. We did not observe a change in survivin and NF-kappaB promoter activity in HER-2 expressing MCF7 cells.

Conclusion: Our results indicate that short term over expression of HER-2 up regulates antiapoptotic proteins Bcl-2 and survivin in MCF7 cells. We determined that survivin is up-regulated via ERK activation and PI3K signalling. Additionally we show that survivin up-regulation is not at transcriptional level. These data provide insight into the mechanism(s) by which induction of HER-2 over expression up-regulates survivin and Bcl-2 and identifies new targets for therapy of breast cancer.

Show MeSH
Related in: MedlinePlus