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. 2011 Mar 24;30(12):1402-12.
doi: 10.1038/onc.2010.521. Epub 2010 Nov 15.

Inhibition of NF-kappa B activity in mammary epithelium increases tumor latency and decreases tumor burden

L Connelly  1 , W Barham, H M Onishko, T Sherrill, L A Chodosh, T S Blackwell, F E Yull
Affiliations

Inhibition of NF-kappa B activity in mammary epithelium increases tumor latency and decreases tumor burden

L Connelly et al. Oncogene. .

Abstract

The transcription factor nuclear factor kappa B (NF-κB) is activated in human breast cancer tissues and cell lines. However, it is unclear whether NF-κB activation is a consequence of tumor formation or a contributor to tumor development. We developed a doxycycline (dox)-inducible mouse model, termed DNMP, to inhibit NF-κB activity specifically within the mammary epithelium during tumor development in the polyoma middle T oncogene (PyVT) mouse mammary tumor model. DNMP females and PyVT littermate controls were treated with dox from 4 to 12 weeks of age. We observed an increase in tumor latency and a decrease in final tumor burden in DNMP mice compared with PyVT controls. A similar effect with treatment from 8 to 12 weeks indicates that outcome is independent of effects on postnatal virgin ductal development. In both cases, DNMP mice were less likely to develop lung metastases than controls. Treatment from 8 to 9 weeks was sufficient to impact primary tumor formation. Inhibition of NF-κB increases apoptosis in hyperplastic stages of tumor development and decreases proliferation at least in part by reducing Cyclin D1 expression. To test the therapeutic potential of NF-κB inhibition, we generated palpable tumors by orthotopic injection of PyVT cells and then treated systemically with the NF-κB inhibitor thymoquinone (TQ). TQ treatment resulted in a reduction in tumor volume and weight as compared with vehicle-treated control. These data indicate that epithelial NF-κB is an active contributor to tumor progression and demonstrate that inhibition of NF-κB could have a significant therapeutic impact even at later stages of mammary tumor progression.

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Figures

Figure 1
Figure 1
Inhibition of NF-κB activity in the PyVT model. A) Expression of NF-κB p65 in nuclear extracts from 7 week and 12 week old wild type and PyVT mammary glands. Protein expression was analyzed by western blot of nuclear extracts. Western blot for TATA binding protein (TBP) was performed as a loading control. Densitometry was performed on Western blots and levels normalized to TBP loading control. Data are represented as mean ± S.E. band density. * indicates p<0.05, significantly different from control; n=3. B) Expression of phosphorylated IκBα in cytoplasmic extracts from wild type mammary glands and PyVT (L129) orthotopic tumors. Western blot of IκB and p42/44 MAPK was performed as loading controls. Expression of myc-tagged DN-IκBα transgene in C) mammary glands (top) or tissues (bottom) from PyVT control and DNMP mice treated with dox (2g/L from 4 to 12 weeks old). Protein expression was analyzed by western blot of whole cell extracts. Western blot for actin was performed as a loading control (n=5). D) Levels of NF-κB p65 in nuclear extracts from PyVT control and DNMP mammary glands treated with dox (2g/L from 8 to 9 weeks old). Protein expression was analyzed by western blot of nuclear extracts. Western blot for TATA binding protein (TBP) was performed as a loading control. Densitometry was performed on Western blots and levels normalized to TBP loading control. Data are represented as mean ± S.E. band density. * indicates p<0.05, significantly different from control; n=4.
Figure 2
Figure 2
An increase in tumor latency and a reduction in tumor burden is observed in DNMP mice treated with dox (2g/L) from 4 to 12 weeks old. A) Mice were palpated twice weekly for formation of mammary gland tumors. Kaplan Meier curves of the time until tumor palpation (p<0.05, significantly different from control; control n= 7, DNMP n=13). Day of dox administration was designated as day zero, day of tumor appearance was calculated from this point. B) Final palpable mammary gland tumor number at 12 weeks. *** indicates p<0.0001, significantly different from control; control n=8, DNMP n=13. C) All mammary glands were collected at 12 weeks and weighed to determine total gland mass (g). *** indicates p<0.0001, significantly different from control; control n=8, DNMP n=13. D) Number of mice per group with at least one surface lung tumor.
Figure 3
Figure 3
An increase in tumor latency and a reduction in tumor burden is observed in DNMP mice treated with dox (2g/L) from 8 to 12 weeks old. A) Whole mount analyses of DNMP and control mice. B) Mice were palpated twice weekly for formation of mammary gland tumors. Kaplan Meier curves show the time until tumor palpation (p<0.05, significantly different from control; control n= 9, DNMP n=7.) Day of dox administration was designated as day zero, day of tumor appearance was calculated from this point. C) Final palpable tumor number at 12 weeks. ** indicates p=0.0082, significantly different from control; control n=9, DNMP n=7. D) All mammary glands were collected at 12 weeks and weighed to determine total gland mass (g). ** indicates p=0.0093, significantly different from control; control n=7, DNMP n=9. E) Number of mice per group with at least one surface lung tumor.
Figure 4
Figure 4
Inhibition of NF-κB signaling leads to increased apoptosis during early stages of tumorigenesis. PyVT controls and DNMP were treated with dox (2g/L) from 8 to 9 weeks old. A) Whole mount analyses. B) TUNEL immunofluorescence staining was performed and quantified according to tumor stage (Hyp = hyperplasia, Ad = adenoma, EC = early carcinoma, LC = late carcinoma). Representative images from hyperplastic lesions (20X) are shown: TUNEL (green) and DAPI (blue). C) F4/80 immunofluorescence staining was performed on hyperplastic lesions in PyVT control and DNMP sections and quantified according to tumor stage. Representative images (40X) are shown: F4/80 (green) and DAPI (blue). TUNEL and F4/80 positive cells were quantified (3 images from each stage) using metamorph software and expressed as a percentage of total cells, * indicates p<0.05, significantly different from control; n=6. D) Myc immunofluorescence staining was performed to detect transgene expression as recognized by the Myc tag. Representative image (20X) of mammary hyperplastic lesions: Myc tag (red) and DAPI (blue).
Figure 5
Figure 5
Inhibition of NF-κB signaling blocks proliferation and reduces Cyclin D1 expression during mammary tumorigenesis. PyVT controls and DNMP were treated with dox (2g/L) from 8 to 9 weeks old. A) Ki67 immunohistochemistry, representative images (63X) are shown. Ki67 positive cells were counted and expressed as a percentage of total cells, * indicates p<0.05; n=3. B) Phospho-histone H3 (PH3) immunofluorescence staining, representative images (20X) are shown: PH3 (green) and DAPI (blue). PH3 positive cells were quantified using Metamorph software and expressed as a percentage of total cells, * indicates p<0.05, significantly different from control; n=6. C) Expression of Cyclin D1 in whole cell mammary gland extracts. Protein expression was analyzed by western blot. Western blot for Actin was performed as a loading control. Densitometry was performed on Western blots and levels normalized to Actin loading control. Data are represented as mean ± S.E. band density. * indicates p<0.05, significantly different from control; n=4.
Figure 6
Figure 6
TQ inhibits NF-κB activity and reduces growth of orthotopic tumors. A) L129 cells were treated with TNF-α (10 ng/ml) for 6 hours in the presence or absence of TQ (50μM). NF-κB activity was measured by luciferase assay. Data are represented as mean ± S.E. luciferase activity. * indicates p<0.05, *** indicates p<0.0001 significantly different; n=3. L129 cells (1 χ 106) were injected into the fat pads of wild type mice and mice were treated daily from day 10 post-injection with IP injections of TQ (40 mg/kg) or vehicle until harvest. B) Tumor volume was measured with calipers every second day. C) Mammary tumors were collected at 10 days after treatment began and weighed to determine total gland mass (g). Representative tumors from control and TQ treatment groups are shown. * indicates p<0.05, significantly different from control; control n=7, TQ n=6.
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