Cytoplasmic localization of proline, glutamic acid, leucinerich protein 1 (PELP1) induces breast epithelial cell migration through up-regulation of inhibitor of κb kinase ϵ and inflammatory cross-talk with macrophages

Brian J. Girard, Todd P Knutson, Bethanie Kuker, Laura McDowell, Kaylee Schwertfeger, Julie H Ostrander

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Abstract

Cytoplasmic localization of proline, glutamic acid, leucinerich protein 1 (PELP1) is observed in ∼40% of women with invasive breast cancer. In mouse models, PELP1 overexpression in the mammary gland leads to premalignant lesions and eventually mammary tumors. In preliminary clinical studies, cytoplasmic localization of PELP1 was seen in 36% of women at high risk of developing breast cancer. Here, we investigated whether cytoplasmic PELP1 signaling promotes breast cancer initiation in models of immortalized human mammary epithelial cells (HMECs). Global gene expression analysis was performed on HMEC lines expressing vector control, PELP1-wt, or mutant PELP1 in which the nuclear localization sequence was altered, resulting in cytoplasmic localization of PELP1 (PELP1-cyto). Global gene expression analysis identified that PELP1-cyto expression in HMECs induced NF-∼B signaling pathways. Western blotting analysis of PELP1-cyto HMECs showed up-regulation of inhibitor of ∼B kinase κ (IKKκ) and increased phosphorylation of the NF-∼B subunit RelB. To determine whether secreted factors produced by PELP1-cyto HMECs promote macrophage activation, THP-1 macrophages were treated with HMEC-conditioned medium (CM). PELP1-cyto CM induced changes in THP-1 gene expression as compared with control cell CM. Double conditioned medium (DCM) from the activated THP-1 cells was then applied to HMECs to determine whether paracrine signaling from PELP1-cyto-activated macrophages could in turn promote migration of HMECs. PELP1-cyto DCM induced robust HMEC migration, which was reduced in DCM from PELP1-cyto HMECs expressing IKKκ shRNA. Our findings suggest that cytoplasmic localization of PELP1 up-regulates pro-tumorigenic IKKκ and secreted inflammatory signals, which through paracrine macrophage activation regulates the migratory phenotype associated with breast cancer initiation.

Original languageEnglish (US)
Pages (from-to)339-350
Number of pages12
JournalJournal of Biological Chemistry
Volume292
Issue number1
DOIs
StatePublished - Jan 6 2017

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Macrophages
Proline
Cell Movement
Glutamic Acid
Breast
Phosphotransferases
Up-Regulation
Epithelial Cells
Proteins
Conditioned Culture Medium
Breast Neoplasms
Gene expression
Macrophage Activation
Gene Expression
Chemical activation
Paracrine Communication
Phosphorylation
Human Mammary Glands
Small Interfering RNA

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@article{e70eb45c2aa3418fa7ffb7ffe49a17df,
title = "Cytoplasmic localization of proline, glutamic acid, leucinerich protein 1 (PELP1) induces breast epithelial cell migration through up-regulation of inhibitor of κb kinase ϵ and inflammatory cross-talk with macrophages",
abstract = "Cytoplasmic localization of proline, glutamic acid, leucinerich protein 1 (PELP1) is observed in ∼40{\%} of women with invasive breast cancer. In mouse models, PELP1 overexpression in the mammary gland leads to premalignant lesions and eventually mammary tumors. In preliminary clinical studies, cytoplasmic localization of PELP1 was seen in 36{\%} of women at high risk of developing breast cancer. Here, we investigated whether cytoplasmic PELP1 signaling promotes breast cancer initiation in models of immortalized human mammary epithelial cells (HMECs). Global gene expression analysis was performed on HMEC lines expressing vector control, PELP1-wt, or mutant PELP1 in which the nuclear localization sequence was altered, resulting in cytoplasmic localization of PELP1 (PELP1-cyto). Global gene expression analysis identified that PELP1-cyto expression in HMECs induced NF-∼B signaling pathways. Western blotting analysis of PELP1-cyto HMECs showed up-regulation of inhibitor of ∼B kinase κ (IKKκ) and increased phosphorylation of the NF-∼B subunit RelB. To determine whether secreted factors produced by PELP1-cyto HMECs promote macrophage activation, THP-1 macrophages were treated with HMEC-conditioned medium (CM). PELP1-cyto CM induced changes in THP-1 gene expression as compared with control cell CM. Double conditioned medium (DCM) from the activated THP-1 cells was then applied to HMECs to determine whether paracrine signaling from PELP1-cyto-activated macrophages could in turn promote migration of HMECs. PELP1-cyto DCM induced robust HMEC migration, which was reduced in DCM from PELP1-cyto HMECs expressing IKKκ shRNA. Our findings suggest that cytoplasmic localization of PELP1 up-regulates pro-tumorigenic IKKκ and secreted inflammatory signals, which through paracrine macrophage activation regulates the migratory phenotype associated with breast cancer initiation.",
author = "Girard, {Brian J.} and Knutson, {Todd P} and Bethanie Kuker and Laura McDowell and Kaylee Schwertfeger and Ostrander, {Julie H}",
year = "2017",
month = "1",
day = "6",
doi = "10.1074/jbc.M116.739847",
language = "English (US)",
volume = "292",
pages = "339--350",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
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T1 - Cytoplasmic localization of proline, glutamic acid, leucinerich protein 1 (PELP1) induces breast epithelial cell migration through up-regulation of inhibitor of κb kinase ϵ and inflammatory cross-talk with macrophages

AU - Girard, Brian J.

AU - Knutson, Todd P

AU - Kuker, Bethanie

AU - McDowell, Laura

AU - Schwertfeger, Kaylee

AU - Ostrander, Julie H

PY - 2017/1/6

Y1 - 2017/1/6

N2 - Cytoplasmic localization of proline, glutamic acid, leucinerich protein 1 (PELP1) is observed in ∼40% of women with invasive breast cancer. In mouse models, PELP1 overexpression in the mammary gland leads to premalignant lesions and eventually mammary tumors. In preliminary clinical studies, cytoplasmic localization of PELP1 was seen in 36% of women at high risk of developing breast cancer. Here, we investigated whether cytoplasmic PELP1 signaling promotes breast cancer initiation in models of immortalized human mammary epithelial cells (HMECs). Global gene expression analysis was performed on HMEC lines expressing vector control, PELP1-wt, or mutant PELP1 in which the nuclear localization sequence was altered, resulting in cytoplasmic localization of PELP1 (PELP1-cyto). Global gene expression analysis identified that PELP1-cyto expression in HMECs induced NF-∼B signaling pathways. Western blotting analysis of PELP1-cyto HMECs showed up-regulation of inhibitor of ∼B kinase κ (IKKκ) and increased phosphorylation of the NF-∼B subunit RelB. To determine whether secreted factors produced by PELP1-cyto HMECs promote macrophage activation, THP-1 macrophages were treated with HMEC-conditioned medium (CM). PELP1-cyto CM induced changes in THP-1 gene expression as compared with control cell CM. Double conditioned medium (DCM) from the activated THP-1 cells was then applied to HMECs to determine whether paracrine signaling from PELP1-cyto-activated macrophages could in turn promote migration of HMECs. PELP1-cyto DCM induced robust HMEC migration, which was reduced in DCM from PELP1-cyto HMECs expressing IKKκ shRNA. Our findings suggest that cytoplasmic localization of PELP1 up-regulates pro-tumorigenic IKKκ and secreted inflammatory signals, which through paracrine macrophage activation regulates the migratory phenotype associated with breast cancer initiation.

AB - Cytoplasmic localization of proline, glutamic acid, leucinerich protein 1 (PELP1) is observed in ∼40% of women with invasive breast cancer. In mouse models, PELP1 overexpression in the mammary gland leads to premalignant lesions and eventually mammary tumors. In preliminary clinical studies, cytoplasmic localization of PELP1 was seen in 36% of women at high risk of developing breast cancer. Here, we investigated whether cytoplasmic PELP1 signaling promotes breast cancer initiation in models of immortalized human mammary epithelial cells (HMECs). Global gene expression analysis was performed on HMEC lines expressing vector control, PELP1-wt, or mutant PELP1 in which the nuclear localization sequence was altered, resulting in cytoplasmic localization of PELP1 (PELP1-cyto). Global gene expression analysis identified that PELP1-cyto expression in HMECs induced NF-∼B signaling pathways. Western blotting analysis of PELP1-cyto HMECs showed up-regulation of inhibitor of ∼B kinase κ (IKKκ) and increased phosphorylation of the NF-∼B subunit RelB. To determine whether secreted factors produced by PELP1-cyto HMECs promote macrophage activation, THP-1 macrophages were treated with HMEC-conditioned medium (CM). PELP1-cyto CM induced changes in THP-1 gene expression as compared with control cell CM. Double conditioned medium (DCM) from the activated THP-1 cells was then applied to HMECs to determine whether paracrine signaling from PELP1-cyto-activated macrophages could in turn promote migration of HMECs. PELP1-cyto DCM induced robust HMEC migration, which was reduced in DCM from PELP1-cyto HMECs expressing IKKκ shRNA. Our findings suggest that cytoplasmic localization of PELP1 up-regulates pro-tumorigenic IKKκ and secreted inflammatory signals, which through paracrine macrophage activation regulates the migratory phenotype associated with breast cancer initiation.

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