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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

Doxycycline-inducible expression of HER-2 protein. Three pTRE-HER2-transfected clones (H1, H2 and H3) and two pTRE-transfected control MCF-7 clones (C1 and C2) were incubated with or without doxycycline. Whole cell protein extracts were collected and evaluated for HER2 expression by western blotting using HER2 antibodies.
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Figure 1: Doxycycline-inducible expression of HER-2 protein. Three pTRE-HER2-transfected clones (H1, H2 and H3) and two pTRE-transfected control MCF-7 clones (C1 and C2) were incubated with or without doxycycline. Whole cell protein extracts were collected and evaluated for HER2 expression by western blotting using HER2 antibodies.

Mentions: The HER-2 cDNA was cloned under the control of tetracycline response element in pTREhygro as described above, to produce the plasmid pTRE-HER2. The MCF7 cell line was transfected with pTRE-HER2, and clones were isolated by selection with hygromycin B and screened for tetracycline inducible expression of HER-2. Doxycyclin-inducible expression of HER-2 was quantified by Western blotting. Three HER-2-transfected clones (H1, H2 and H3) and two vector-transfected control clones (C1 and C2) were incubated with or without doxycycline. Whole cell protein extracts were prepared and evaluated for ErbB2 expression by western blotting (Figure 1). Induction of HER-2 was quantified by NIH Java image processing program ImageJ. In the presence of Dox HER-2 was increased 4.1, 2.7 and 2.8 fold compared to without Dox in H1, H2 and H3 clones respectively. No significant difference in HER-2 with or without Dox was found in the vector control clones (C1 and C2).


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)

Doxycycline-inducible expression of HER-2 protein. Three pTRE-HER2-transfected clones (H1, H2 and H3) and two pTRE-transfected control MCF-7 clones (C1 and C2) were incubated with or without doxycycline. Whole cell protein extracts were collected and evaluated for HER2 expression by western blotting using HER2 antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Doxycycline-inducible expression of HER-2 protein. Three pTRE-HER2-transfected clones (H1, H2 and H3) and two pTRE-transfected control MCF-7 clones (C1 and C2) were incubated with or without doxycycline. Whole cell protein extracts were collected and evaluated for HER2 expression by western blotting using HER2 antibodies.
Mentions: The HER-2 cDNA was cloned under the control of tetracycline response element in pTREhygro as described above, to produce the plasmid pTRE-HER2. The MCF7 cell line was transfected with pTRE-HER2, and clones were isolated by selection with hygromycin B and screened for tetracycline inducible expression of HER-2. Doxycyclin-inducible expression of HER-2 was quantified by Western blotting. Three HER-2-transfected clones (H1, H2 and H3) and two vector-transfected control clones (C1 and C2) were incubated with or without doxycycline. Whole cell protein extracts were prepared and evaluated for ErbB2 expression by western blotting (Figure 1). Induction of HER-2 was quantified by NIH Java image processing program ImageJ. In the presence of Dox HER-2 was increased 4.1, 2.7 and 2.8 fold compared to without Dox in H1, H2 and H3 clones respectively. No significant difference in HER-2 with or without Dox was found in the vector control clones (C1 and C2).

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