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. 2009 Jan;171(1):9-21.
doi: 10.1667/RR1472.1.

NF-kappaB-mediated HER2 overexpression in radiation-adaptive resistance

Ning Cao  1 , Shiyong Li, Zhaoqing Wang, Kazi Mokim Ahmed, Michael E Degnan, Ming Fan, Joseph R Dynlacht, Jian Jian Li
Affiliations

NF-kappaB-mediated HER2 overexpression in radiation-adaptive resistance

Ning Cao et al. Radiat Res. 2009 Jan.

Abstract

The molecular mechanisms governing acquired tumor resistance during radiotherapy remain to be elucidated. In breast cancer patients, overexpression of HER2 (human epidermal growth factor receptor 2) is correlated with aggressive tumor growth and increased recurrence. In the present study, we demonstrate that HER2 expression can be induced by radiation in breast cancer cells with a low basal level of HER2. Furthermore, HER2-postive tumors occur at a much higher frequency in recurrent invasive breast cancer (59%) compared to the primary tumors (41%). Interestingly, NF-kappaB is required for radiation-induced HER2 transactivation. HER2 was found to be co-activated with basal and radiation-induced NF-kappaB activity in radioresistant but not radiosensitive breast cancer cell lines after long-term radiation exposure, indicating that NF-kappaB-mediated HER2 overexpression is involved in radiation-induced repopulation in heterogeneous tumors. Finally, we found that inhibition of HER2 resensitizes the resistant cell lines to radiation. Since HER2 is shown to activate NF-kappaB, our data suggest a loop-like HER2-NF-kappaB-HER2 pathway in radiation-induced adaptive resistance in breast cancer cells.

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Figures

FIG. 1
FIG. 1
HER2 expression was induced in breast cancer cells by γ radiation. Panels A–C: HER2 mRNA levels were increased in MDA-MB-231 (panel A) and MCF-7 (panel B) breast cancer cells but not in MCF-7/HER2 cells (panel C). Total RNA purified from cells 24 h after exposure to 5 Gy of γ rays (n = 3; mean ± SE; **P < 0.01). Panels D and E: HER2 protein levels were enhanced in irradiated (5 Gy γ rays) MDA-MB-231 and MCF-7 cells (panel D) as well as in the radioresistant population that survived long-term fractionated irradiation (MCF+FIR; panel E) (21) but not in HER2-overexpressing MCF-7/HER2 cells measured by Western blot analysis. Panel F: HER2 expression was induced in irradiated MDA-MB-231 xenograft tumors (3 × 2 Gy; total tumor dose 6 Gy) detected by HER2 immunohistochemistry (red) with DAPI nuclear staining (blue) 24 h after irradiation (T1 = sham-irradiated control; additional HER2 immunohistochemistry data can be found in Supplementary Fig. S1). Panel G: Western blot of HER2 in MDA-MB-231 control (C) and irradiated xenografts 24 h after irradiation. Panel H: Increased frequency of HER2-positive breast cancer cells in human recurrent invasive breast cancers compared to primary tumors analyzed by immunohistochemistry (IHC) and FISH.
FIG. 2
FIG. 2
Gamma-radiation exposure enhanced the recruitment of NF-κB to HER2 promoter. Panel A: Schematic of the HER2 promoter locus of two fragments (A and B) studied by ChIP assays. Panel B: Chromatin of control and irradiated MCF-7 and MDA-MB-231 cells was immunoprecipitated with antibodies to p65, p50, p52 and c-Rel or mouse IgG. Both fragments A and B were amplified by PCR, and total chromatin (total input) was positive control for PCR. The PCR fragment of the IκBα promoter region (−1134/−902) and GAPDH were also included as positive and negative controls, respectively. Panel C: Sham-irradiated and γ-irradiated MDA-MB-231 xenografts were examined for recruitment of p65 and p50 to the HER2 promoter (fragment A) and IκBα promoter (NF-κB positive control) by ChIP assays. Fragment B l was included as the negative control. Panel D: MCF-7 cells were preincubated with 2 µM NF-κB inhibitor, IMD-0354 or DMSO (as solvent control) for 5 h or were transiently transfected with mutant IκBα before exposure to 5 Gy or sham irradiation. Recruitment of p65 and p50 to the HER2 promoter (fragment A) and IκBα promoter (NF-κB positive control) were examined by ChIP assays. Panel E: The reduction of binding of NF-κB to the HER2 and IκBα promoters by different inhibitors was estimated by densitometry normalized to the input band.
FIG. 3
FIG. 3
NF-κB is required for radiation-induced HER2 expression. Panel A: MCF-7 and MDA-MB-231 cells transfected with NF-κB-driven luciferase reporters were treated with IMD-0354 (2 µM) or DMSO for 5 h before exposure to 5 Gy or sham irradiation. Luciferase activities were determined 24 h after irradiation (n = 3; mean ± SE; **P < 0.01). Results are shown as the percentage of the value for untreated MCF-7 cells. Panel B: pGL2 luciferase plasmid (V), HER2-controlled luciferase reporter (HER2), or HER2-controlled luciferase reporter with deleted NF-κB binding site (mHER2) was transfected into MCF-7 and MDA-MB-231 cells. Luciferase activity was measured 24 h after exposure to 5 Gy or sham irradiation (n = 3; mean ± SE; **P < 0.01). Panels C and D: MCF-7 and MDA-MB-231 cells transfected with HER2 luciferase reporters were treated with IMD-0354 (2 µM) or DMSO for 5 h before exposure to 5 Gy or sham irradiation. Luciferase activities were determined 24 h after irradiation (panel C; n = 3; mean ± SE; **P < 0.01). Panel D: Western blot analysis of HER2 levels 24 h after irradiation. Panels E and F: Western blot analysis of MCF-7 cells treated with scramble siRNA or p65 siRNA for 60 h (panel E; lip = transfectants reagent only; scr = 20 nM scrambled siRNA; siRNA = p65 siRNA), and luciferase activity was measured 24 h after irradiation. Results are shown as a percentage of the value for untreated MCF-7 cells (panel F; n = 3; mean ± SE; **P < 0.01).
FIG. 4
FIG. 4
NF-κB-mediated HER2 expression is associated with the survival of different MDA-MB-231 cell populations after radiation exposure. Panel A: Western blot of HER2 expression in sham-irradiated MDA-MB-231 cells (sham) and in cells of six cloned cell lines isolated from the MDA+FIR cell population. Panel B: NF-κB transactivation of sham-irradiated MDA-MB-231 cells (sham) and in cells of six cloned MDA+FIR cell lines with or without exposure to 5 Gy γ rays. The luciferase activities are shown as a percentage of the value for sham-irradiated cells. Panel C: Clonogenic survival of sham-irradiated MDA-MB-231 cells (sham) and cells of six cloned MDA+FIR cell lines irradiated with of γ rays (inset: correlation between HER2 protein levels and clonogenicity of MDA+FIR cell lines; n = 3; mean ± SE).
FIG. 5
FIG. 5
Radiosensitization of HER2-overexpressing cells by NF-κB inhibition. Panels A and B: Inhibition of HER2 overexpression by IMD-0354 (2 µM for 5 h) in MCF+IR and radioresistant (C4, C5) cells but not in MCF-7/HER2 cells and relatively radiosensitive (C2) MDA+IR cells. Panels C–E: Clonogenic survival of cells of MDA+IR cell lines C2 (panel C), C4 (panel D), and C5 (panel E) pretreated with the NF-κB inhibitor IMD-0354 (2 µM, 5 h) or DMSO before radiation exposure (n = 3; mean ± SE; **P < 0.01 compared to DMSO-treated cells). Panel F: Inhibition of HER2 expression in cells of the radioresistant MDA+FIR cell lines C2 and C4 by NF-κB p65 siRNA. Panels G and H: Clonogenic survival of cells of the radioresistant MDA+FIR cell lines C2 (panel G) and C4 (panel H) treated with NF-κB p65 siRNA (20 nM for 60 h before irradiation; n = 3; mean ± SE; **P < 0.01).
FIG. 6
FIG. 6
Radiosensitization by HER2 siRNA. Panel A: Inhibition of HER2 expression in MCF+IR and MCF-7/HER2 cells by 20 nM HER2 siRNA (lip =transfection reagent; scr =20 nM scrambled siRNA; siRNA = HER2 siRNA). Panels B and C: Clonogenic survival of MCF-7/HER2 (panel B) and MCF+IR (panel C) cells treated with HER2 siRNA (20 nM for 60 h) and then irradiated (points, mean; n = 3; bars, SE; **P < 0.01, *P < 0.05 compared to the scramble siRNA control). Panel D: Inhibition of HER2 expression in radioresistant (C4, C5) and radiosensitive (C2) MDA+FIR cells treated with HER2 siRNA or scrambled siRNA (20 nM for 60 h). Panels E–G: Clonogenic survival of radiosensitive C2 (panel E) and radioresistant C4 (panel F) and C5 (panel G) cells treated with HER2 siRNA (20 nM for 60 h before irradiation; n = 3; mean ± SE; **P < 0.01). Panel H: Schematic representation of radiation-induced loop-like HER2-NF-κB-HER2 pathway in radiation-induced adaptive resistance. IR, radiation.
FIG. 6
FIG. 6
Radiosensitization by HER2 siRNA. Panel A: Inhibition of HER2 expression in MCF+IR and MCF-7/HER2 cells by 20 nM HER2 siRNA (lip =transfection reagent; scr =20 nM scrambled siRNA; siRNA = HER2 siRNA). Panels B and C: Clonogenic survival of MCF-7/HER2 (panel B) and MCF+IR (panel C) cells treated with HER2 siRNA (20 nM for 60 h) and then irradiated (points, mean; n = 3; bars, SE; **P < 0.01, *P < 0.05 compared to the scramble siRNA control). Panel D: Inhibition of HER2 expression in radioresistant (C4, C5) and radiosensitive (C2) MDA+FIR cells treated with HER2 siRNA or scrambled siRNA (20 nM for 60 h). Panels E–G: Clonogenic survival of radiosensitive C2 (panel E) and radioresistant C4 (panel F) and C5 (panel G) cells treated with HER2 siRNA (20 nM for 60 h before irradiation; n = 3; mean ± SE; **P < 0.01). Panel H: Schematic representation of radiation-induced loop-like HER2-NF-κB-HER2 pathway in radiation-induced adaptive resistance. IR, radiation.
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References

    1. Kim JJ, Tannock IF. Repopulation of cancer cells during therapy: an important cause of treatment failure. Nat. Rev. Cancer 5. 2005;5:516–525. - PubMed
    1. Morgan WF. Genomic instability and bystander effects: a paradigm shift in radiation biology? Mil. Med. 2002;167:44–45. - PubMed
    1. Bisht KS, Bradbury CM, Mattson D, Kaushal A, Sowers A, Markovina S, Ortiz KL, Sieck LK, Isaacs JS, Gius D. Geldanamycin and 17-allylamino-17-demethoxygeldanamycin potentiate the in vitro and in vivo radiation response of cervical tumor cells via the heat shock protein 90-mediated intracellular signaling and cytotoxicity. Cancer Res. 2003;63:8984–8995. - PubMed
    1. Amundson SA, Grace MB, McLeland CB, Epperly MW, Yeager A, Zhan Q, Greenberger JS, Fornace AJ., Jr Human in vivo radiation-induced biomarkers: gene expression changes in radiotherapy patients. Cancer Res. 2004;64:6368–6371. - PubMed
    1. Dewey WC, Ling CC, Meyn RE. Radiation-induced apoptosis: relevance to radiotherapy. Int. J. Radiat. Oncol. Biol. Phys. 1995;33:781–796. - PubMed

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