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Background Selective estrogen receptor modulators (SERMs) such as Tamoxifen (TAM) can significantly improve breast cancer-specific survival for women with ER-positive (ER+) disease. However, resistance to TAM remains a major clinical problem. The resistant phenotype is usually not driven by loss or mutation of ER; instead, changes in multiple proliferative and/or survival pathways override the inhibitory effects of TAM. Estrogen-related receptor gamma (ERRγ) is an orphan member of the nuclear receptor superfamily that promotes TAM resistance in ER+ breast cancer cells. In this study, we sought to clarify the mechanism(s) by which this orphan nuclear receptor is regulated and, in turn, affects TAM resistance. Methods mRNA and protein expression/phosphorylation were monitored by RT-PCR and Western blotting, respectively. Site-directed mutagenesis was used to disrupt consensus ERK target sites. Cell proliferation and cell cycle progression were measured by flow cytometric methods. ERRγ transcriptional activity was assessed by dual-luciferase promoter-reporter assays. Results We show that ERRγ protein levels are affected by the activation state of ERK/MAPK, and mutation of consensus ERK target sites impairs ERRγ-driven transcriptional activity and TAM resistance. Conclusions These findings shed new light on the functional significance of ERRγ in ER+ breast cancer, and are the first to demonstrate a role for kinase regulation of this orphan nuclear receptor.

Transfer of a phosphoryl group from autophosphorylated CheA (P-CheA) to CheY is an important step in the bacterial chemotaxis signal transduction pathway. This reaction involves CheY (i) binding to the P2 domain of P-CheA and then (ii) acquiring the phosphoryl group from the P1 domain. Crystal structures indicated numerous side chain interactions at the CheY-P2 binding interface. To investigate the individual contributions of the P2 side chains involved in these contacts, we analyzed the effects of eight alanine substitution mutations on CheA-CheY binding interactions. An F214A substitution in P2 caused ϳ1,000-fold reduction in CheA-CheY binding affinity, while Ala substitutions at other P2 positions had small effects (E171A, E178A, and I216A) or no detectable effects (H181A, D202A, D207A, and C213A) on binding affinity. These results are discussed in relation to previous in silico predictions of hot-spot and anchor positions at the CheA-CheY interface. We also investigated the consequences of these mutations for chemotaxis signal transduction in living cells. CheA(F214A) was defective in mediating localization of CheY-YFP to the large clusters of signaling proteins that form at the poles of Escherichia coli cells, while the other CheA variants did not differ from wild-type (wt) CheA (CheA wt ) in this regard. In our set of mutants, only CheA(F214A) exhibited a markedly diminished ability to support chemotaxis in motility agar assays. Surprisingly, however, in FRET assays that monitored receptor-regulated production of phospho-CheY, CheA(F214A) (and each of the other Ala substitution mutants) performed just as well as CheA wt . Overall, our findings indicate that F214 serves as an anchor residue at the CheA-CheY interface and makes an important contribution to the binding energy in vitro and in vivo; however, loss of this contribution does not have a large negative effect on the overall ability of the signaling pathway to modulate P-CheY levels in response to chemoattractants.Chemotaxis in Escherichia coli and numerous other bacterial species involves regulation of the level of phosphorylated CheY (P-CheY) in response to spatial gradients of beneficial and harmful chemicals. P-CheY plays a crucial role in chemotaxis by enabling cells to control how frequently they change directions as they swim (2,48,58,63). The level of P-CheY in a cell reflects the relative rates of phosphorylation (mediated by CheA) and dephosphorylation (mediated by CheZ) (15,46). CheA functions as an autokinase, and this activity is regulated by membrane-spanning receptor proteins responsible for binding chemical ligands that serve as attractants or repellents (7, 16). Autophosphorylated CheA (P-CheA) serves as a phosphodonor for CheY, and the P-CheY generated by this interaction can bind to the switch component of the flagellar motor, inducing changes in cell swimming direction by promoting changes in the direction of flagellar rotation (41,65,66). This sequence of events provides a signal transduction pathway that allows the chemotaxis re...

Low linear energy transfer (low-LET) γ-ray exposure is a risk factor for colorectal cancer (CRC). Due to high-LET nature, energetic iron ions are expected to pose greater CRC risks to astronauts undertaking long duration space missions beyond low earth orbit (LEO). Wild-type p53-induced phosphatase 1 (Wip1) is important for cellular DNA damage response and its abrogation is reported to inhibit spontaneous intestinal tumorigenesis in APCMin/+ mice, a well-studied mouse model of human CRC. However, Wip1 in relation to radiation-induced especially energetic iron ions-induced intestinal tumorigenesis has not been investigated in APCMin/+ mice. While we earlier demonstrated greater intestinal tumorigenic potential of energetic iron ions relative to 137Cs γ rays, the purpose of the current study was to investigate if Wip1 abrogation could influence radiation quality dependent intestinal tumorigenesis in APCMin/+ mice. Intestinal tumor frequency and grade was assessed in APCMin/+;Wip1−/− mice and results were compared to those in APCMin/+;Wip1+/+ mice after exposure to a mean absorbed dose of 2 Gy from 137Cs γ rays or 1.6 Gy from 1 GeV/n iron ions. Cellular differentiation and proliferation were also assessed in the intestinal tumors of irradiated and sham irradiated mice. Decreased tumor frequency and lower tumor grade was observed in APCMin/+;Wip1−/− relative to APCMin/+;Wip1+/+ mice. Notably, similar decrease (~6 fold in both groups) in tumor number was observed in sham-, and γ-irradiated APCMin/+;Wip1−/− relative to APCMin/+;Wip1+/+ mice. However, tumorigenesis in energetic iron ions group was reduced ~8 fold in APCMin/+;Wip1−/− relative to APCMin/+;Wip1+/+ mice. Significantly lower proliferation/differentiation index in tumors of energetic iron ions exposed APCMin/+;Wip1−/− relative to APCMin/+;Wip1+/+ mice suggests reduced proliferation and enhanced differentiation as a result of Wip1 abrogation. In conclusion, the current study demonstrates that the absence of Wip1 could block radiation-induced intestinal tumorigenesis irrespective of radiation quality and has implications for developing preventive strategies against tumorigenic potential of radiation exposure on earth and in outer space.

Selective estrogen receptor modulators (SERMs) such as Tamoxifen (TAM) can significantly improve breast cancer-specific survival for women with ER-positive (ER+) disease. However, resistance to TAM remains a major clinical problem. Estrogen-related receptor gamma (ERRγ) is an orphan nuclear receptor with broad, structural similarities to classical ER that is widely implicated in the transcriptional regulation of energy homeostasis. We previously reported that ERRγ is upregulated during the acquisition of TAM resistance in ER+ breast cancer cell lines, exogenous expression of ERRγ is sufficient to induce TAM resistance, and ERRγ mRNA is significantly increased in tumor samples from women with ER+ breast cancer who relapse following TAM treatment. Because ERRγ has no known ligand, our recent studies have focused on understanding how the expression and activity of this orphan nuclear receptor is regulated, and how this contributes to the TAM resistant phenotype. We have found that TAM-resistant breast cancer cells in which endogenous ERRγ is upregulated show a concomitant hyper activation of p44/p42 ERK. Furthermore, ERK activity (but not that of JNK or p38 MAPK) directly enhances ERRγ protein stability via Serines 57, 81, and/or 219 of the receptor. Phospho-deficient ERRγ is impaired in its ability to induce TAM resistance, and this is associated with a significant reduction in transcriptional activity at the estrogen-related response element (ERRE) half-site, in particular. This led us to hypothesize that ERRγ action at ERREs is most relevant to the development of TAM resistance. We examined a meta-list of validated, ERRE-containing ERR target genes in association with distant metastasis-free survival (DMFS) within 5 years of primary diagnosis in each of 3 publicly available clinical datasets comprised of ER+ breast cancer patients treated with TAM, and obtained a list of 37 differentially expressed targets. The proximal promoter regions of these 37 genes are enriched for binding sites for ELK1, a well-known ERK substrate. Sub-network analysis reveals enrichment of genes whose protein products regulate the mitochondrial unfolded protein response (UPRmito). We also performed differential dependency network (DDN) analysis in an independent dataset using the full meta-list of ERRγ target genes and also identified genes associated with UPRmito. These data suggest a potentially novel role for ERK-mediated regulation of ERRγ in mitochondrial protein folding in TAM-resistant breast cancer. Citation Format: Rebecca B. Riggins, Mary M. Heckler, Hemang Thakor, Cara C. Schafer, Salendra Singh, Ye Tian, Yuriy Gusev, Subha Madhavan, Yue Wang. Phospho-dependent regulation of ERRγ expression, transcriptional activity, and Tamoxifen resistance in ER+ breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3570. doi:10.1158/1538-7445.AM2013-3570