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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

Transgenic mouse model targets expression of cIKKβ specifically to mammary epithelium.a Diagram shows crossing of two transgenic strains necessary to generate the double transgenic (*/*) IKMV mouse model with doxycycline inducible transgene expression. Littermates lacking either one or both transgenes (*/-, −/*, or −/−) were used throughout our studies as littermate controls. For characterization, IKMV and control littermates were treated with doxycycline (2 g/L) for 3 days and mammary tissue collected for the following assays: b RT-PCR of whole mammary homogenates confirms the FLAG-tagged transgene is dox-inducible. Upon dox-treatment, the transgene was expressed in the */* double transgenic IKMV animals, but absent in dox-treated, single transgenic control mice (−/*). Double transgenic */* IKMV mice that did not receive dox-treatment showed no detectable transgene expression. c TransAM ELISA assay using IKMV and control mammary nuclear homogenates shows that nuclear p65 in IKMV samples actively binds the NF-κB DNA consensus sequence (n = 4 control, n = 4 IKMV samples; **p = 0.0069)
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Fig1: Transgenic mouse model targets expression of cIKKβ specifically to mammary epithelium.a Diagram shows crossing of two transgenic strains necessary to generate the double transgenic (*/*) IKMV mouse model with doxycycline inducible transgene expression. Littermates lacking either one or both transgenes (*/-, −/*, or −/−) were used throughout our studies as littermate controls. For characterization, IKMV and control littermates were treated with doxycycline (2 g/L) for 3 days and mammary tissue collected for the following assays: b RT-PCR of whole mammary homogenates confirms the FLAG-tagged transgene is dox-inducible. Upon dox-treatment, the transgene was expressed in the */* double transgenic IKMV animals, but absent in dox-treated, single transgenic control mice (−/*). Double transgenic */* IKMV mice that did not receive dox-treatment showed no detectable transgene expression. c TransAM ELISA assay using IKMV and control mammary nuclear homogenates shows that nuclear p65 in IKMV samples actively binds the NF-κB DNA consensus sequence (n = 4 control, n = 4 IKMV samples; **p = 0.0069)

Mentions: Previously, our group has studied the activation of NF-κB in mammary tissue in vivo using IκBα knock-out mice [15]. In these transgenics, deletion of the inhibitor is systemic and activity through the canonical pathway is increased within every tissue, causing mortality by day 9 post birth [25]. However, transplant of mammary tissue from 6 day old female pups into wild type donors allowed us to observe the effects of NF-κB activation during pubertal mammary gland development. Using this model, we found an increase in lateral ductal branching and pervasive intraductal hyperplasia in the IκBα knock-out reconstituted glands. This was the first indication that aberrant NF-κB activation could lead to dramatic changes in ductal growth. As in most mammary transplant methods, stromal and epithelial components were co-transplanted into recipients. Because IκBα had been deleted in both of these components, it was impossible to determine whether it was the epithelial derived NF-κB activation that caused the resulting phenotype. To address this and to enable specific temporal regulation of the increased activation of NF-κB, we developed a doxycycline (dox) inducible model which would target activation specifically to mammary epithelium. This model requires two transgenic components: tet-O-cIKKβ mice are combined with MMTV-rtTA transgenics to produce double transgenic mice that we have termed “IKMV” (Fig. 1a). RT-PCR of whole mammary homogenates confirms the FLAG-tagged cIKKβ transgene is dox-inducible. Upon dox-treatment, transgene expression was evident in the */* double transgenic IKMV mammary, but absent in dox-treated, single transgenic control mice (−/*). Double transgenic */* IKMV mice that did not receive dox-treatment showed no detectable transgene expression (Fig. 1b). Thus, in all subsequent studies, “IKMV” refers to the double transgenic mice and “control” refers to littermates lacking one or both transgenes, which behave as wild type mice. To confirm the ability of the transgene to activate NF-κB activity, TransAM ELISA was completed using the nuclear fraction of mammary tissue lysates. This showed that there is increased binding of nuclear p65 to the NF-κB DNA consensus sequence following transgene induction (Fig. 1c).Fig. 1


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)

Transgenic mouse model targets expression of cIKKβ specifically to mammary epithelium.a Diagram shows crossing of two transgenic strains necessary to generate the double transgenic (*/*) IKMV mouse model with doxycycline inducible transgene expression. Littermates lacking either one or both transgenes (*/-, −/*, or −/−) were used throughout our studies as littermate controls. For characterization, IKMV and control littermates were treated with doxycycline (2 g/L) for 3 days and mammary tissue collected for the following assays: b RT-PCR of whole mammary homogenates confirms the FLAG-tagged transgene is dox-inducible. Upon dox-treatment, the transgene was expressed in the */* double transgenic IKMV animals, but absent in dox-treated, single transgenic control mice (−/*). Double transgenic */* IKMV mice that did not receive dox-treatment showed no detectable transgene expression. c TransAM ELISA assay using IKMV and control mammary nuclear homogenates shows that nuclear p65 in IKMV samples actively binds the NF-κB DNA consensus sequence (n = 4 control, n = 4 IKMV samples; **p = 0.0069)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Transgenic mouse model targets expression of cIKKβ specifically to mammary epithelium.a Diagram shows crossing of two transgenic strains necessary to generate the double transgenic (*/*) IKMV mouse model with doxycycline inducible transgene expression. Littermates lacking either one or both transgenes (*/-, −/*, or −/−) were used throughout our studies as littermate controls. For characterization, IKMV and control littermates were treated with doxycycline (2 g/L) for 3 days and mammary tissue collected for the following assays: b RT-PCR of whole mammary homogenates confirms the FLAG-tagged transgene is dox-inducible. Upon dox-treatment, the transgene was expressed in the */* double transgenic IKMV animals, but absent in dox-treated, single transgenic control mice (−/*). Double transgenic */* IKMV mice that did not receive dox-treatment showed no detectable transgene expression. c TransAM ELISA assay using IKMV and control mammary nuclear homogenates shows that nuclear p65 in IKMV samples actively binds the NF-κB DNA consensus sequence (n = 4 control, n = 4 IKMV samples; **p = 0.0069)
Mentions: Previously, our group has studied the activation of NF-κB in mammary tissue in vivo using IκBα knock-out mice [15]. In these transgenics, deletion of the inhibitor is systemic and activity through the canonical pathway is increased within every tissue, causing mortality by day 9 post birth [25]. However, transplant of mammary tissue from 6 day old female pups into wild type donors allowed us to observe the effects of NF-κB activation during pubertal mammary gland development. Using this model, we found an increase in lateral ductal branching and pervasive intraductal hyperplasia in the IκBα knock-out reconstituted glands. This was the first indication that aberrant NF-κB activation could lead to dramatic changes in ductal growth. As in most mammary transplant methods, stromal and epithelial components were co-transplanted into recipients. Because IκBα had been deleted in both of these components, it was impossible to determine whether it was the epithelial derived NF-κB activation that caused the resulting phenotype. To address this and to enable specific temporal regulation of the increased activation of NF-κB, we developed a doxycycline (dox) inducible model which would target activation specifically to mammary epithelium. This model requires two transgenic components: tet-O-cIKKβ mice are combined with MMTV-rtTA transgenics to produce double transgenic mice that we have termed “IKMV” (Fig. 1a). RT-PCR of whole mammary homogenates confirms the FLAG-tagged cIKKβ transgene is dox-inducible. Upon dox-treatment, transgene expression was evident in the */* double transgenic IKMV mammary, but absent in dox-treated, single transgenic control mice (−/*). Double transgenic */* IKMV mice that did not receive dox-treatment showed no detectable transgene expression (Fig. 1b). Thus, in all subsequent studies, “IKMV” refers to the double transgenic mice and “control” refers to littermates lacking one or both transgenes, which behave as wild type mice. To confirm the ability of the transgene to activate NF-κB activity, TransAM ELISA was completed using the nuclear fraction of mammary tissue lysates. This showed that there is increased binding of nuclear p65 to the NF-κB DNA consensus sequence following transgene induction (Fig. 1c).Fig. 1

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