Limits...
Aberrant activation of NF-κB signaling in mammary epithelium leads to abnormal growth and ductal carcinoma in situ.

Barham W, Chen L, Tikhomirov O, Onishko H, Gleaves L, Stricker TP, Blackwell TS, Yull FE - BMC Cancer (2015)

Bottom Line: We found that even a short pulse of NF-κB activation could induce profound remodeling of mammary ductal structures.These results indicate that aberrant NF-κB activation within mammary epithelium can lead to molecular and morphological changes consistent with the earliest stages of breast cancer.Thus, inhibition of NF-κB signaling following acute inflammation or the initial signs of hyperplastic ductal growth could represent an important opportunity for breast cancer prevention.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Vanderbilt University Medical Center, 23rd Ave S and Pierce PRB 325, Nashville, TN, 37232, USA. Whitney.Barham@vanderbilt.edu.

ABSTRACT

Background: Approximately 1 in 5 women diagnosed with breast cancer are considered to have in situ disease, most often termed ductal carcinoma in situ (DCIS). Though recognized as a risk factor for the development of more invasive cancer, it remains unclear what factors contribute to DCIS development. It has been shown that inflammation contributes to the progression of a variety of tumor types, and nuclear factor kappa B (NF-κB) is recognized as a master-regulator of inflammatory signaling. However, the contributions of NF-κB signaling to tumor initiation are less well understood. Aberrant up-regulation of NF-κB activity, either systemically or locally within the breast, could occur due to a variety of commonly experienced stimuli such as acute infection, obesity, or psychological stress. In this study, we seek to determine if activation of NF-κB in mammary epithelium could play a role in the formation of hyperplastic ductal lesions.

Methods: Our studies utilize a doxycycline-inducible transgenic mouse model in which constitutively active IKKβ is expressed specifically in mammary epithelium. All previously published models of NF-κB modulation in the virgin mammary gland have been constitutive models, with transgene or knock-out present throughout the life and development of the animal. For the first time, we will induce activation at later time points after normal ducts have formed, thus being able to determine if NF-κB activation can promote pre-malignant changes in previously normal mammary epithelium.

Results: We found that even a short pulse of NF-κB activation could induce profound remodeling of mammary ductal structures. Short-term activation created hyperproliferative, enlarged ducts with filled lumens. Increased expression of inflammatory markers was concurrent with the down-regulation of hormone receptors and markers of epithelial differentiation. Furthermore, the oncoprotein mucin 1, known to be up-regulated in human and mouse DCIS, was over-expressed and mislocalized in the activated ductal tissue.

Conclusions: These results indicate that aberrant NF-κB activation within mammary epithelium can lead to molecular and morphological changes consistent with the earliest stages of breast cancer. Thus, inhibition of NF-κB signaling following acute inflammation or the initial signs of hyperplastic ductal growth could represent an important opportunity for breast cancer prevention.

No MeSH data available.


Related in: MedlinePlus

Aberrant activation of NF-κB in mammary epithelium induces downstream signaling consistent with early tumorigenesis. RNA was isolated from mammary glands of IKMV and control mice after 3 days of dox treatment (6 week time point). a qRT-PCR for a variety of gene targets revealed increased expression of inflammatory markers as well as decreased expression of hormone receptors and markers of epithelial differentiation (bars represent log fold change of IKMV vs. control; p values for each comparison can be found in Table 2. b Flow cytometry indicates there is an increased percentage of CD45+/F4/80+ macrophages in mammary glands following NF-κB activation, and c immunohistochemistry reveals that F4/80 positive macrophages have infiltrated the mammary ducts of IKMV mice (positive cells are dark brown). d Immunofluorescent staining reveals decreased nuclear localization of ERα in IKMV ducts (red; dense and nuclear in control epithelium)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4590702&req=5

Fig7: Aberrant activation of NF-κB in mammary epithelium induces downstream signaling consistent with early tumorigenesis. RNA was isolated from mammary glands of IKMV and control mice after 3 days of dox treatment (6 week time point). a qRT-PCR for a variety of gene targets revealed increased expression of inflammatory markers as well as decreased expression of hormone receptors and markers of epithelial differentiation (bars represent log fold change of IKMV vs. control; p values for each comparison can be found in Table 2. b Flow cytometry indicates there is an increased percentage of CD45+/F4/80+ macrophages in mammary glands following NF-κB activation, and c immunohistochemistry reveals that F4/80 positive macrophages have infiltrated the mammary ducts of IKMV mice (positive cells are dark brown). d Immunofluorescent staining reveals decreased nuclear localization of ERα in IKMV ducts (red; dense and nuclear in control epithelium)

Mentions: Following sacrifice, mammary glands #2-4 were harvested for analysis. Lymph nodes of the #4 glands were removed prior to collection. Glands were minced and placed in 3 mL’s of DMEM/F12 containing 3 mg/mL of Collagenase A (Roche) and 100 units/mL Hyaluronidase (Sigma). Glands were incubated in digestion media overnight at 4 °C, followed by 2 h of incubation at 37 °C the following morning. After digestion, cells were pelleted and the fatty layer at the top of the supernatant was discarded. After straining cells through a 70 micron filter, red blood cells were lysed using ACK buffer. Remaining cells were then washed and counted using a hemocytometer. Cells were blocked with anti- mouse CD16/CD32 antibody (eBioscience) before staining with anti-mouse antibodies: CD45 (30-F11) (eBioscience) and F4/80 (BM8) (Life Technologies). DAPI nuclear stain was used to determine viability. Analysis was performed on an LSRII cytometer with DIVA software (BD Biosciences). Gating strategy can be found in Additional file 1. Values for the graph in Fig. 7b were obtained by taking the total number of CD45+F4/80+ positive cells for each sample and dividing that value by the total number of viable cells in the sample (DAPI negative).


Aberrant activation of NF-κB signaling in mammary epithelium leads to abnormal growth and ductal carcinoma in situ.

Barham W, Chen L, Tikhomirov O, Onishko H, Gleaves L, Stricker TP, Blackwell TS, Yull FE - BMC Cancer (2015)

Aberrant activation of NF-κB in mammary epithelium induces downstream signaling consistent with early tumorigenesis. RNA was isolated from mammary glands of IKMV and control mice after 3 days of dox treatment (6 week time point). a qRT-PCR for a variety of gene targets revealed increased expression of inflammatory markers as well as decreased expression of hormone receptors and markers of epithelial differentiation (bars represent log fold change of IKMV vs. control; p values for each comparison can be found in Table 2. b Flow cytometry indicates there is an increased percentage of CD45+/F4/80+ macrophages in mammary glands following NF-κB activation, and c immunohistochemistry reveals that F4/80 positive macrophages have infiltrated the mammary ducts of IKMV mice (positive cells are dark brown). d Immunofluorescent staining reveals decreased nuclear localization of ERα in IKMV ducts (red; dense and nuclear in control epithelium)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4590702&req=5

Fig7: Aberrant activation of NF-κB in mammary epithelium induces downstream signaling consistent with early tumorigenesis. RNA was isolated from mammary glands of IKMV and control mice after 3 days of dox treatment (6 week time point). a qRT-PCR for a variety of gene targets revealed increased expression of inflammatory markers as well as decreased expression of hormone receptors and markers of epithelial differentiation (bars represent log fold change of IKMV vs. control; p values for each comparison can be found in Table 2. b Flow cytometry indicates there is an increased percentage of CD45+/F4/80+ macrophages in mammary glands following NF-κB activation, and c immunohistochemistry reveals that F4/80 positive macrophages have infiltrated the mammary ducts of IKMV mice (positive cells are dark brown). d Immunofluorescent staining reveals decreased nuclear localization of ERα in IKMV ducts (red; dense and nuclear in control epithelium)
Mentions: Following sacrifice, mammary glands #2-4 were harvested for analysis. Lymph nodes of the #4 glands were removed prior to collection. Glands were minced and placed in 3 mL’s of DMEM/F12 containing 3 mg/mL of Collagenase A (Roche) and 100 units/mL Hyaluronidase (Sigma). Glands were incubated in digestion media overnight at 4 °C, followed by 2 h of incubation at 37 °C the following morning. After digestion, cells were pelleted and the fatty layer at the top of the supernatant was discarded. After straining cells through a 70 micron filter, red blood cells were lysed using ACK buffer. Remaining cells were then washed and counted using a hemocytometer. Cells were blocked with anti- mouse CD16/CD32 antibody (eBioscience) before staining with anti-mouse antibodies: CD45 (30-F11) (eBioscience) and F4/80 (BM8) (Life Technologies). DAPI nuclear stain was used to determine viability. Analysis was performed on an LSRII cytometer with DIVA software (BD Biosciences). Gating strategy can be found in Additional file 1. Values for the graph in Fig. 7b were obtained by taking the total number of CD45+F4/80+ positive cells for each sample and dividing that value by the total number of viable cells in the sample (DAPI negative).

Bottom Line: We found that even a short pulse of NF-κB activation could induce profound remodeling of mammary ductal structures.These results indicate that aberrant NF-κB activation within mammary epithelium can lead to molecular and morphological changes consistent with the earliest stages of breast cancer.Thus, inhibition of NF-κB signaling following acute inflammation or the initial signs of hyperplastic ductal growth could represent an important opportunity for breast cancer prevention.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Vanderbilt University Medical Center, 23rd Ave S and Pierce PRB 325, Nashville, TN, 37232, USA. Whitney.Barham@vanderbilt.edu.

ABSTRACT

Background: Approximately 1 in 5 women diagnosed with breast cancer are considered to have in situ disease, most often termed ductal carcinoma in situ (DCIS). Though recognized as a risk factor for the development of more invasive cancer, it remains unclear what factors contribute to DCIS development. It has been shown that inflammation contributes to the progression of a variety of tumor types, and nuclear factor kappa B (NF-κB) is recognized as a master-regulator of inflammatory signaling. However, the contributions of NF-κB signaling to tumor initiation are less well understood. Aberrant up-regulation of NF-κB activity, either systemically or locally within the breast, could occur due to a variety of commonly experienced stimuli such as acute infection, obesity, or psychological stress. In this study, we seek to determine if activation of NF-κB in mammary epithelium could play a role in the formation of hyperplastic ductal lesions.

Methods: Our studies utilize a doxycycline-inducible transgenic mouse model in which constitutively active IKKβ is expressed specifically in mammary epithelium. All previously published models of NF-κB modulation in the virgin mammary gland have been constitutive models, with transgene or knock-out present throughout the life and development of the animal. For the first time, we will induce activation at later time points after normal ducts have formed, thus being able to determine if NF-κB activation can promote pre-malignant changes in previously normal mammary epithelium.

Results: We found that even a short pulse of NF-κB activation could induce profound remodeling of mammary ductal structures. Short-term activation created hyperproliferative, enlarged ducts with filled lumens. Increased expression of inflammatory markers was concurrent with the down-regulation of hormone receptors and markers of epithelial differentiation. Furthermore, the oncoprotein mucin 1, known to be up-regulated in human and mouse DCIS, was over-expressed and mislocalized in the activated ductal tissue.

Conclusions: These results indicate that aberrant NF-κB activation within mammary epithelium can lead to molecular and morphological changes consistent with the earliest stages of breast cancer. Thus, inhibition of NF-κB signaling following acute inflammation or the initial signs of hyperplastic ductal growth could represent an important opportunity for breast cancer prevention.

No MeSH data available.


Related in: MedlinePlus