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

Many cells within aberrant ducts are epithelial, transgene-expressing, and have high levels of NF-κB activation. 6 week old virgin IKMV and control littermates were dox-treated (2 g/L) for 3 days prior to sacrifice. a Immunofluorescent staining of control and IKMV tissue reveals that IKMV ducts are filled with CK8/18 positive luminal epithelium. CK5 and SMA were used as markers of basal/myoepithelium. b Separate staining shows that the FLAG-tagged cIKKβ transgene is expressed by cells within the IKMV hyperplastic ducts (red, FLAG stain). In addition, high magnification images of ductal tissue from IKMV and control littermates confirmed that the transgene is localized appropriately within the cytoplasm of IKMV mammary epithelium and is driving concurrent nuclear localization of phospho-p65 (green)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: Many cells within aberrant ducts are epithelial, transgene-expressing, and have high levels of NF-κB activation. 6 week old virgin IKMV and control littermates were dox-treated (2 g/L) for 3 days prior to sacrifice. a Immunofluorescent staining of control and IKMV tissue reveals that IKMV ducts are filled with CK8/18 positive luminal epithelium. CK5 and SMA were used as markers of basal/myoepithelium. b Separate staining shows that the FLAG-tagged cIKKβ transgene is expressed by cells within the IKMV hyperplastic ducts (red, FLAG stain). In addition, high magnification images of ductal tissue from IKMV and control littermates confirmed that the transgene is localized appropriately within the cytoplasm of IKMV mammary epithelium and is driving concurrent nuclear localization of phospho-p65 (green)

Mentions: Upon observing such a dramatic filling of the IKMV ducts, we endeavored to determine whether the cells within the ducts were epithelial. To do this, we completed immunofluorescent staining for the luminal epithelial marker cytokeratin 8/18 (CK8/18). This revealed that many of the cells filling the lumens stain positive for this marker (Fig. 4a). In addition, FLAG-tagged transgene expression was found throughout the aberrant ducts in IKMV glands (Fig. 4b). Since transgene expression is specific to MMTV-rtTA expressing mammary epithelial cells in the IKMV system, this again suggests that many of the cells filling the ducts are epithelial. Finally, we wanted to confirm that the transgene expression was indeed driving NF-κB activation within the epithelium at the 3 day time point. Using immunofluorescent staining, and high magnification images, we observed cytoplasmic localization of the transgene within mammary epithelial cells resulting in robust nuclear localization of phospho-p65 (ser 536) (Fig. 4b).Fig. 4


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)

Many cells within aberrant ducts are epithelial, transgene-expressing, and have high levels of NF-κB activation. 6 week old virgin IKMV and control littermates were dox-treated (2 g/L) for 3 days prior to sacrifice. a Immunofluorescent staining of control and IKMV tissue reveals that IKMV ducts are filled with CK8/18 positive luminal epithelium. CK5 and SMA were used as markers of basal/myoepithelium. b Separate staining shows that the FLAG-tagged cIKKβ transgene is expressed by cells within the IKMV hyperplastic ducts (red, FLAG stain). In addition, high magnification images of ductal tissue from IKMV and control littermates confirmed that the transgene is localized appropriately within the cytoplasm of IKMV mammary epithelium and is driving concurrent nuclear localization of phospho-p65 (green)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: Many cells within aberrant ducts are epithelial, transgene-expressing, and have high levels of NF-κB activation. 6 week old virgin IKMV and control littermates were dox-treated (2 g/L) for 3 days prior to sacrifice. a Immunofluorescent staining of control and IKMV tissue reveals that IKMV ducts are filled with CK8/18 positive luminal epithelium. CK5 and SMA were used as markers of basal/myoepithelium. b Separate staining shows that the FLAG-tagged cIKKβ transgene is expressed by cells within the IKMV hyperplastic ducts (red, FLAG stain). In addition, high magnification images of ductal tissue from IKMV and control littermates confirmed that the transgene is localized appropriately within the cytoplasm of IKMV mammary epithelium and is driving concurrent nuclear localization of phospho-p65 (green)
Mentions: Upon observing such a dramatic filling of the IKMV ducts, we endeavored to determine whether the cells within the ducts were epithelial. To do this, we completed immunofluorescent staining for the luminal epithelial marker cytokeratin 8/18 (CK8/18). This revealed that many of the cells filling the lumens stain positive for this marker (Fig. 4a). In addition, FLAG-tagged transgene expression was found throughout the aberrant ducts in IKMV glands (Fig. 4b). Since transgene expression is specific to MMTV-rtTA expressing mammary epithelial cells in the IKMV system, this again suggests that many of the cells filling the ducts are epithelial. Finally, we wanted to confirm that the transgene expression was indeed driving NF-κB activation within the epithelium at the 3 day time point. Using immunofluorescent staining, and high magnification images, we observed cytoplasmic localization of the transgene within mammary epithelial cells resulting in robust nuclear localization of phospho-p65 (ser 536) (Fig. 4b).Fig. 4

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