Cancer Stem Cell Phenotypes in ER+ Breast Cancer Models Are Promoted by PELP1/AIB1 Complexes.

Proline, glutamic acid, leucine-rich protein 1 (PELP1) is overexpressed in approximately 80% of invasive breast tumors. PELP1 dynamically shuttles between the nucleus and cytoplasm, but is primarily nuclear in normal breast tissue. However, altered localization of PELP1 to the cytoplasm is an oncogenic event that promotes breast cancer initiation and progression. Herein, interacting partners unique to cytoplasmic PELP1 and the mechanisms by which these interactions promote oncogenic PELP1 signaling were sought. AIB1 (amplified in breast cancer 1; also known as SRC-3 or NCOA3) was identified as a novel binding partner of cytoplasmic PELP1 in both estrogen receptor-positive (ER+) and ER-negative cell lines. Cytoplasmic PELP1 expression elevated basal phosphorylation levels (i.e., activation) of AIB1 at Thr24, enhanced ALDH+ tumorsphere formation, and upregulated specific target genes independently of hormone stimulation. Direct manipulation of AIB1 levels using shRNA abrogated cytoplasmic PELP1-induced tumorsphere formation and downregulated cytoplasmic PELP1-specific target genes. SI-2, an AIB1 inhibitor, limited the PELP1/AIB1 interaction and decreased cytoplasmic PELP1-induced tumorsphere formation. Similar results were observed in a murine-derived MMTV-AIB1 tumor cell line. Furthermore, in vivo syngeneic tumor studies revealed that PELP1 knockdown resulted in increased survival of tumor-bearing mice as compared with mice injected with control cells.Implications: These data demonstrate that cytoplasmic PELP1/AIB1-containing complexes function to promote advanced cancer phenotypes, including outgrowth of stem-like cells, associated with estrogen-independent breast cancer progression. Mol Cancer Res; 16(4); 707-19. ©2018 AACR.

Cytoplasmic Localization of Proline, Glutamic Acid, Leucine-rich Protein 1 (PELP1) Induces Breast Epithelial Cell Migration through Up-regulation of Inhibitor of κB Kinase ϵ and Inflammatory Cross-talk with Macrophages.

Cytoplasmic localization of proline, glutamic acid, leucine-rich 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-κB signaling pathways. Western blotting analysis of PELP1-cyto HMECs showed up-regulation of inhibitor of κB kinase ϵ (IKKϵ) and increased phosphorylation of the NF-κB 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 IKKϵ shRNA. Our findings suggest that cytoplasmic localization of PELP1 up-regulates pro-tumorigenic IKKϵ and secreted inflammatory signals, which through paracrine macrophage activation regulates the migratory phenotype associated with breast cancer initiation.

Cytoplasmic PELP1 and ERRgamma protect human mammary epithelial cells from Tam-induced cell death.

Tamoxifen (Tam) is the only FDA-approved chemoprevention agent for pre-menopausal women at high risk for developing breast cancer. While Tam reduces a woman's risk of developing estrogen receptor positive (ER+) breast cancer, the molecular mechanisms associated with risk reduction are poorly understood. Prior studies have shown that cytoplasmic proline, glutamic acid and leucine rich protein 1 (PELP1) promotes Tam resistance in breast cancer cell lines. Herein, we tested for PELP1 localization in breast epithelial cells from women at high risk for developing breast cancer and found that PELP1 was localized to the cytoplasm in 36% of samples. In vitro, immortalized HMECs expressing a nuclear localization signal (NLS) mutant of PELP1 (PELP1-cyto) were resistant to Tam-induced death. Furthermore, PELP1-cyto signaling through estrogen-related receptor gamma (ERRγ) promoted cell survival in the presence of Tam. Overexpression of ERRγ in immortalized HMECs protected cells from Tam-induced death, while knockdown of ERRγ sensitized PELP1-cyto expressing HMECs to Tam. Moreover, Tam-induced HMEC cell death was independent of apoptosis and involved accumulation of the autophagy marker LC3-II. Expression of PELP1-cyto and ERRγ reduced Tam-induced LC3-II accumulation, and knockdown of ERRγ increased LC3-II levels in response to Tam. Additionally, PELP1-cyto expression led to the upregulation of MMP-3 and MAOB, known PELP1 and ERRγ target genes, respectively. Our data indicate that cytoplasmic PELP1 induces signaling pathways that converge on ERRγ to promote cell survival in the presence of Tam. These data suggest that PELP1 localization and/or ERRγ activation could be developed as tissue biomarkers for Tam responsiveness.

PELP1: a review of PELP1 interactions, signaling, and biology.

Proline, glutamic acid, and leucine rich protein 1 (PELP1) is a large multi-domain protein that has been shown to modulate an increasing number of pathways and biological processes. The first reports describing the cloning and characterization of PELP1 showed that it was an estrogen receptor coactivator. PELP1 has now been shown to be a coregulator for a growing number of transcription factors. Furthermore, recent reports have shown that PELP1 is a member of chromatin remodeling complexes. In addition to PELP1 nuclear functions, it has been shown to have cytoplasmic signaling functions as well. In the cytoplasm PELP1 acts as a scaffold molecule and mediates rapid signaling from growth factor and hormone receptors. PELP1 signaling ultimately plays a role in cancer biology by increasing proliferation and metastasis, among other cellular processes. Here we will review (1) the cloning and characterization of PELP1 expression, (2) interacting proteins, (3) PELP1 signaling, and (4) PELP1-mediated biology.

Breast tumor kinase (Brk/PTK6) is a mediator of hypoxia-associated breast cancer progression.

Basal-type triple-negative breast cancers (TNBC) are aggressive and difficult to treat relative to luminal-type breast cancers. TNBC often express abundant Met receptors and are enriched for transcriptional targets regulated by hypoxia-inducible factor-1α (HIF-1α), which independently predict cancer relapse and increased risk of metastasis. Brk/PTK6 is a critical downstream effector of Met signaling and is required for hepatocyte growth factor (HGF)-induced cell migration. Herein, we examined the regulation of Brk by HIFs in TNBC in vitro and in vivo. Brk mRNA and protein levels are upregulated strongly in vitro by hypoxia, low glucose, and reactive oxygen species. In HIF-silenced cells, Brk expression relied upon both HIF-1α and HIF-2α, which we found to regulate BRK transcription directly. HIF-1α/2α silencing in MDA-MB-231 cells diminished xenograft growth and Brk reexpression reversed this effect. These findings were pursued in vivo by crossing WAP-Brk (FVB) transgenic mice into the MET(Mut) knockin (FVB) model. In this setting, Brk expression augmented MET(Mut)-induced mammary tumor formation and metastasis. Unexpectedly, tumors arising in either MET(Mut) or WAP-Brk × MET(Mut) mice expressed abundant levels of Sik, the mouse homolog of Brk, which conferred increased tumor formation and decreased survival. Taken together, our results identify HIF-1α/2α as novel regulators of Brk expression and suggest that Brk is a key mediator of hypoxia-induced breast cancer progression. Targeting Brk expression or activity may provide an effective means to block the progression of aggressive breast cancers.