Lrig1 is the founding member of the “Lrig” family and has been implicated in the negative regulation of several oncogenic receptor tyrosine kinases including ErbB2. Lrig1 is expressed at low levels in several cancer types but is over-expressed in some prostate and colorectal tumors. Given this heterogeneity, whether Lrig1 functions to suppress or promote tumor growth remains a critical question. Previously, we found that Lrig1 was poorly expressed in ErbB2-positive breast cancer, suggesting that Lrig1 has a growth inhibitory role in this tumor type. However, breast cancer is a complex disease, with ErbB2-positive tumors accounting for just 25% of all breast cancers. To gain a better understanding of the role of Lrig1 in breast cancer, we examined its expression in estrogen receptor alpha (ERα)-positive disease which accounts for the majority of breast cancers. We find that Lrig1 is expressed at significantly higher levels in ERα-positive disease as compared to ERα-negative disease. Our study provides a molecular rationale for Lrig1 enrichment in ERα-positive disease by demonstrating that Lrig1 is a target of ERα. Estrogen stimulates Lrig1 accumulation and disruption of this induction enhances estrogen-dependent tumor cell growth, suggesting that Lrig1 functions as an estrogen regulated growth suppressor. Additionally, we find that Lrig1 expression correlates with prolonged relapse-free survival in ERα-positive breast cancer, identifying Lrig1 as a new prognostic marker in this setting. Finally, we demonstrate that ErbB2 activation antagonizes ERα-driven Lrig1 expression, providing a mechanistic explanation for Lrig1 loss in ErbB2-positive breast cancer. This work provides strong evidence for a growth inhibitory role for Lrig1 in breast cancer.

The ErbB3 receptor tyrosine kinase contributes to a variety of developmental processes, and its overexpression and aberrant activation promote tumor progression and therapeutic resistance. Accumulating evidence suggests that tumor overexpression may be mediated by the loss of posttranscriptional negative regulatory mechanisms, such as protein degradation, that normally keep receptor levels in check. Our previous studies indicate that the RING finger E3 ubiquitin ligase Nrdp1, a protein lost in breast and other tumor types, suppresses ErbB3 levels by mediating ligand-independent receptor ubiquitination and degradation. Here we demonstrate that Nrdp1 preferentially associates with the nascent form of ErbB3 to accelerate its degradation, and we show that the two proteins colocalize at the endoplasmic reticulum (ER). Blocking the exit of ErbB3 from the ER does not affect the ability of Nrdp1 to mediate receptor ubiquitination or degradation, while functional disruption of the conserved ER-associated degradation (ERAD) pathway ATPase VCP/p97 leads to the Nrdp1-dependent accumulation of ubiquitinated ErbB3 but blocks receptor degradation. Further evidence indicates that the ErbB3 targeted by Nrdp1 for degradation is properly folded and fully functional. Collectively, these observations point to a novel mechanism of receptor tyrosine kinase quantity control wherein steadystate levels of signaling-competent receptor are dictated by an ER-localized degradation pathway.

The LRIG (leucine-rich repeats and immunoglobulin-like domains) family of transmembrane proteins contains three vertebrate members (LRIG1, LRIG2, LRIG3) and one member each in flies (Lambik) and worms (Sma-10). LRIGs have stepped into the spotlight as essential regulators of growth factor receptors, including receptor tyrosine and serine/threonine kinases. LRIGs have been found to both negatively (LRIG1, LRIG3) and positively (Sma-10, LRIG3) regulate growth factor receptor expression and signaling, although the precise molecular mechanisms by which LRIGs function are not yet understood. The most is known about LRIG1, which was recently demonstrated to be a tumor suppressor. Indeed, in vivo experiments reinforce the essential link between LRIG1 and repression of its targets for tissue homeostasis. LRIG1 has also been identified as a stem cell marker and regulator of stem cell quiescence in a variety of tissues, discussed within. Comparably less is known about LRIG2 and LRIG3 although studies to date suggest that their functions are largely distinct from LRIG1 and that they likely do not serve as growth/tumor suppressors. Finally, the translational applications of expressing soluble forms of LRIG1 in LRIG1-deficient tumors are being explored and hold tremendous promise.

LRIG1, a member of the LRIG family of transmembrane leucine rich repeat-containing proteins, is a negative regulator of receptor tyrosine kinase signaling and a tumor suppressor. LRIG1 expression is broadly decreased in human cancer and in breast cancer, low expression of LRIG1 has been linked to decreased relapse-free survival. Recently, low expression of LRIG1 was revealed to be an independent risk factor for breast cancer metastasis and death. These findings suggest that LRIG1 may oppose breast cancer cell motility and invasion, cellular processes which are fundamental to metastasis. However, very little is known of LRIG1 function in this regard. In this study, we demonstrate that LRIG1 is down-regulated during epithelial to mesenchymal transition (EMT) of human mammary epithelial cells, suggesting that LRIG1 expression may represent a barrier to EMT. Indeed, depletion of endogenous LRIG1 in human mammary epithelial cells expands the stem cell population, augments mammosphere formation and accelerates EMT. Conversely, expression of LRIG1 in highly invasive Basal B breast cancer cells provokes a mesenchymal to epithelial transition accompanied by a dramatic suppression of tumorsphere formation and a striking loss of invasive growth in three-dimensional culture. LRIG1 expression perturbs multiple signaling pathways and represses markers and effectors of the mesenchymal state. Furthermore, LRIG1 expression in MDA-MB-231 breast cancer cells significantly slows their growth as tumors, providing the first in vivo evidence that LRIG1 functions as a growth suppressor in breast cancer.

BackgroundThe ZNF217 gene, encoding a C2H2 zinc finger protein, is located at 20q13 and found amplified and overexpressed in greater than 20% of breast tumors. Current studies indicate ZNF217 drives tumorigenesis, yet the regulatory mechanisms of ZNF217 are largely unknown. Because ZNF217 associates with chromatin modifying enzymes, we postulate that ZNF217 functions to regulate specific gene signaling networks. Here, we present a large-scale functional genomic analysis of ZNF217, which provides insights into the regulatory role of ZNF217 in MCF7 breast cancer cells.ResultsChIP-seq analysis reveals that the majority of ZNF217 binding sites are located at distal regulatory regions associated with the chromatin marks H3K27ac and H3K4me1. Analysis of ChIP-seq transcription factor binding sites shows clustering of ZNF217 with FOXA1, GATA3 and ERalpha binding sites, supported by the enrichment of corresponding motifs for the ERalpha-associated cis-regulatory sequences. ERalpha expression highly correlates with ZNF217 in lysates from breast tumors (n = 15), and ERalpha co-precipitates ZNF217 and its binding partner CtBP2 from nuclear extracts. Transcriptome profiling following ZNF217 depletion identifies differentially expressed genes co-bound by ZNF217 and ERalpha; gene ontology suggests a role for ZNF217-ERalpha in expression programs associated with ER+ breast cancer studies found in the Molecular Signature Database. Data-mining of expression data from breast cancer patients correlates ZNF217 with reduced overall survival.ConclusionsOur genome-wide ZNF217 data suggests a functional role for ZNF217 at ERalpha target genes. Future studies will investigate whether ZNF217 expression contributes to aberrant ERalpha regulatory events in ER+ breast cancer and hormone resistance.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-520) contains supplementary material, which is available to authorized users.

◥ miR-127 is downregulated in breast cancer, where it has been shown to suppress the proliferation, migration, and invasion of breast cancer cells. In triple-negative breast cancer (TNBC), miR-127 downregulation correlates with decreased disease-free and overall patient survival. Tumor suppressor miRNAs may hold therapeutic promise but progress has been limited by several factors, including the lability and high cost of miRNA mimics. Here, we take a novel approach to produce a miR-127 prodrug (miR-127 PD ), which we demonstrate is processed to mature, functional miR-127-3p in TNBC tumor cells. miR-127 PD decreased the viability and motility of TNBC cells, sensitized TNBC cells to chemotherapy, and restricted the TNBC stem cell population. Furthermore, systemic delivery of miR-127 PD suppressed tumor growth of MDA-MB-231 and MDA-MB-468 TNBC cells and spontaneous metastasis of MDA-MB-231 cells. In addition, CERK, NANOS1, FOXO6, SOX11, SOX12, FASN, and SUSD2 were identified as novel, functionally important targets of miR-127. In conclusion, our study demonstrates that miR-127 functions as a tumor and metastasis suppressor in TNBC and that delivery of miR-127 may hold promise as a novel therapy.Significance: Exogenous administration of miR-127, which is functionally activated in target cells, inhibits growth and spontaneous metastasis of triple-negative breast cancer.

Background:The Tbx3 transcription factor is over-expressed in breast cancer, where it has been implicated in proliferation, migration and regulation of the cancer stem cell population. The mechanisms that regulate Tbx3 expression in cancer have not been fully explored. In this study, we demonstrate that Tbx3 is repressed by the tumour suppressor miR-206 in breast cancer cells.Methods:Bioinformatics prediction programmes and luciferase reporter assays were used to demonstrate that miR-206 negatively regulates Tbx3. We examined the impact of miR-206 on Tbx3 expression in breast cancer cells using miR-206 mimic and inhibitor. Gene/protein expression was examined by quantitative reverse-transcription–PCR and immunoblotting. The effects of miR-206 and Tbx3 on apoptosis, proliferation, invasion and cancer stem cell population was investigated by cell-death detection, colony formation, 3D-Matrigel and tumorsphere assays.Results:In this study, we examined the regulation of Tbx3 by miR-206. We demonstrate that Tbx3 is directly repressed by miR-206, and that this repression of Tbx3 is necessary for miR-206 to inhibit breast tumour cell proliferation and invasion, and decrease the cancer stem cell population. Moreover, Tbx3 and miR-206 expression are inversely correlated in human breast cancer. Kaplan–Meier analysis indicates that patients exhibiting a combination of high Tbx3 and low miR-206 expression have a lower probability of survival when compared with patients with low Tbx3 and high miR-206 expression. These studies uncover a novel mechanism of Tbx3 regulation and identify a new target of the tumour suppressor miR-206.Conclusions:The present study identified Tbx3 as a novel target of tumour suppressor miR-206 and characterised the miR-206/Tbx3 signalling pathway, which is involved in proliferation, invasion and maintenance of the cancer stem cell population in breast cancer cells. Our results suggest that restoration of miR-206 in Tbx3-positive breast cancer could be exploited for therapeutic benefit.

ErbB3, a member of the ErbB family of receptor tyrosine kinases, is a potent activator of phosphatidyl inositol-3 kinase (PI3K) and mTOR signaling, driving tumor cell survival and therapeutic resistance in breast cancers. In luminal breast cancers, ErbB3 upregulation following treatment with the anti-estrogen fulvestrant enhances PI3K/mTOR-mediated cell survival. However, the mechanism by which ErbB3 is upregulated in fulvestrant-treated cells is unknown. We found that ErbB3 protein levels and cell surface presentation were increased following fulvestrant treatment, focusing our attention on proteins that regulate ErbB3 at the cell surface, including Nrdp1, NEDD4, and LRIG1. Among these, only LRIG1 correlated positively with ERα, but inversely with ErbB3 in clinical breast cancer datasets. LRIG1, an estrogen-inducible ErbB down-regulator, was decreased in a panel of fulvestrant-treated luminal breast cancer cells. Ectopic LRIG1 expression from an estrogen-independent promoter uncoupled LRIG1 from estrogen regulation, thus sustaining LRIG1 and maintaining low ErbB3 levels in fulvestrant-treated cells. An LRIG1 mutant lacking the ErbB3 interaction motif was insufficient to down-regulate ErbB3. Importantly, LRIG1 overexpression improved fulvestrant-mediated growth inhibition, while cells expressing the LRIG1 mutant were poorly sensitive to fulvestrant, despite effective ERα down-regulation. Consistent with these results, LRIG1 expression correlated positively with increased disease-free survival in anti-estrogen-treated breast cancer patients. These data suggest that ERα-dependent expression of LRIG1 dampens ErbB3 signaling in luminal breast cancer cells, and by blocking ERα activity with fulvestrant, LRIG1 is decreased thus permitting ErbB3 accumulation, enhanced ErbB3 signaling to cell survival pathways, and blunting therapeutic response to fulvestrant.