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. 2009 May;174(5):1910-20.
doi: 10.2353/ajpath.2009.080706. Epub 2009 Apr 6.

Nuclear factor-kappaB enhances ErbB2-induced mammary tumorigenesis and neoangiogenesis in vivo

Manran Liu  1 , Xiaoming Ju, Nicole E Willmarth, Mathew C Casimiro, John Ojeifo, Toshiyuki Sakamaki, Sanjay Katiyar, Xuanmao Jiao, Vladimir M Popov, Zuoren Yu, Kongming Wu, David Joyce, Chenguang Wang, Richard G Pestell
Affiliations

Nuclear factor-kappaB enhances ErbB2-induced mammary tumorigenesis and neoangiogenesis in vivo

Manran Liu et al. Am J Pathol. 2009 May.

Abstract

The (HER2/Neu) ErbB2 oncogene is commonly overexpressed in human breast cancer and is sufficient for mammary tumorigenesis in transgenic mice. Nuclear factor (NF)-kappaB activity is increased in both human and murine breast tumors. The immune response to mammary tumorigenesis may regulate tumor progression. The role of endogenous mammary epithelial cell NF-kappaB had not previously been determined in immune-competent animals. Furthermore, the role of the NF-kappaB components, p50 and p65, in tumor growth was not known. Herein, the expression of a stabilized form of the NF-kappaB-inhibiting IkappaBalpha protein (IkappaBalphaSR) in breast tumor cell lines that express oncogenic ErbB2 inhibited DNA synthesis and growth in both two- and three-dimensional cultures. Either NF-kappaB inhibition or selective silencing of p50 or p65 led to a loss of contact-independent tumor growth in vitro. IkappaBalphaSR reversed the features of the oncogene-induced phenotype under three-dimensional growth conditions. The NF-kappaB blockade inhibited ErbB2-induced mammary tumor growth in both immune-competent and immune-deficient mice. These findings were associated with both reduced tumor microvascular density and a reduction in the amount of vascular endothelial growth factor. The expression of IkappaBalphaSR in breast cancer tumors inhibited angiogenesis. Thus, mammary epithelial cell NF-kappaB activity enhances ErbB2-mediated mammary tumorigenesis in vivo by promoting both growth and survival signaling via the promotion of tumor vasculogenesis.

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Figures

Figure 1
Figure 1
ErbB2 induces NF-κB activity. NF-κB activity was assessed using a multimeric NF-κB binding site (3xRel-luc) luciferase reporter and a NF-κB binding assay. A: MCF-7 cells were transfected with either an expression vector for an activating ErbB2* mutant (NeuT) or a control plasmid. 3xRel-luc reporter activity was reduced by the NF-κB inhibitor Bay 11-7082 (left) or transfection with an expression vector encoding IκBαSR (right). B: MCF10A cells, transiently transfected with an expression vector for ErbB2*, were treated with the NF-κB inhibitor Bay 11-7082 (left) or stably transduced with an expression vector for ErbB2* and an expression vector for IκBαSR, relative luciferase activity was determined (right). C and D: NF-κB binding activity (C) or luciferase reporter activity (D) was determined in SKBr3 and NAFA cells stably transduced with an IκBαSR expression vector. Data are relative luciferase activities (mean ± SEM of n > five separate transfections).
Figure 2
Figure 2
The NF-κB components p50 and p65 govern ErbB2-induced cellular proliferation. A: Western blot of MCF10A/ErbB2* (NeuT) cells transduced with p65 or p50 siRNA. MCF10A control or MCF10A cells stably expressing ErbB2* were transduced with siRNA and Western blot analysis was conducted 48 hours after transduction. Western blot analysis was conducted with antibodies as indicated in the figure. B and C: Cellular proliferation assays of p50 or p65 siRNA-transduced MCF10A cell lines were conducted using either MTT assay (B) or cell counting (C). The MCF10A cell lines were stably transfected with expression vectors encoding ErbB2* (NeuT) or control vector (Vec) and either PCMV-IκBαSR or the parental control vector (GFP). The cells were plated in equal number and daily MTT assays conducted as described in the Materials and Methods. The data are shown as mean ± SEM of three separate experiments. *P < 0.05; **P < 0.01.
Figure 3
Figure 3
Endogenous NF-κB maintains breast cancer epithelial cell contact-independent growth and DNA synthesis. Cell cycle analyses by flow cytometry and colony formation assays were conducted in three breast cancer cell lines stably transduced with a retroviral expression vector encoding IκBαSR or a control vector. The data were shown as mean ± SEM for n > three separate experiments. A and C: Western blot for IκBα in NAFA (A) MCF10A, and MCF10A-ErbB2* (C). B and D: Cell proliferation determined by MTT assay (left), cell-cycle distribution assessed by FACS (middle), and colony formation assayed by counting the number of colonies (right). *P < 0.05; **P < 0.01.
Figure 4
Figure 4
NF-κB is required for ErbB2-induced disruption of mammary acinar growth in 3D culture. A and C: MCF10A/ErbB2* (NeuT) cells (A) or NAFA (C) cells transduced with IκBαSR-IRES-GFP (A and C, bottom) or control GFP vector (A and C, top) were grown in 3D Matrigel culture. Immunofluorescence and phase contrast microscopy shows the multilobular structure. Inhibition of NF-κB activity with IκBαSR reverts the transformed morphology to the spherical morphology observed with nontransformed mammary epithelial cells. The volume of MCF10A/ErbB2* (NeuT) (B) and NAFA (D) colonies grown in 3D is reduced 60 to 80% by expression of IκBαSR. E and G: H&E staining of MCF10A/ErbB2* (E) and NAFA (G) cell lines grown in 3D Matrigel demonstrate the solid ball of GFP control, and hollow ball of IκBαSR-expressing cells with (F and H) the relative number shown as mean data. I and J: Immunohistochemical staining for p50 of MCF10A/ErbB2* and NAFA cells. *P < 0.05; **P < 0.01.
Figure 5
Figure 5
NF-κB is required for ErbB2-induced mammary tumor growth in immune-competent and immunodeficient mice. A: FLAG epitope of the IκBαSR protein. B: Tumor volume throughout 4 weeks after implantation of IκBαSR-expressing and control NAFA cells in the FVB strain of mice (n = 12 per group). The data are shown as mean ± SEM. Arrows represent mammary tumor. C: NAFA cells transduced with IκBαSR or control vector were implanted into nude mice. Representative examples of tumor size are shown (left), and tumor growth curves conducted of NAFA cells expressing IκBαSR or control vector in nude mice (n = 20 per group) (right). D: Representative Western blot analysis of NAFA cell-derived tumors transduced with the IκBαSR expression vector or control vector in three separate nude mice. *P < 0.05; **P < 0.01.
Figure 6
Figure 6
Vasculogenesis in ErbB2-induced mammary tumors is NF-κB-dependent. A: Analysis of ErbB2*-induced mammary tumor (NAFA) expressing control vector (GFP) or IκBαSR (n = 5 per group) in nude mice to determine microvessel density. B: Histopathology by H&E staining (left) or immunostaining with CD31 antibody to demarcate tumor blood vessels (right) to which (arrows) point. C: Immunohistochemical staining for candidate pro-angiogenic factors (left) and quantitation of immunohistochemical staining for pro-angiogenic factors, shown as mean ± SEM (right). D: Angiogenesis assay conducted using human umbilical vein endothelial cells treated with media from either control vector or IκBαSR-transduced NAFA cells (left) and quantitation of vessel formation by branch number and length (right). *P < 0.05; **P < 0.01. Original magnifications, ×40.
Figure 7
Figure 7
NF-κB-regulated protein secretion. A: The relative abundance of secreted proteins was determined in the supernatant of NAFA versus NAFA-IκBαSR cells using cytokine arrays. B: Supernatant was analyzed from an equal number of cells after 24 hours of culture. Reduced abundance indicates suppressed by IκBαSR. The data are mean ± SEM of three separate experiments.
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