Aberrant promoter methylation contributes to LRIG1 silencing in basal/triple-negative breast cancer.

BACKGROUND: LRIG1, the founding member of the LRIG (leucine-rich repeat and immunoglobulin-like domain) family of transmembrane proteins, is a negative regulator of receptor tyrosine kinases and a tumour suppressor. Decreased LRIG1 expression is consistently observed in cancer, across diverse tumour types, and is linked to poor patient prognosis. However, mechanisms by which LRIG1 is repressed are not fully understood. Silencing of LRIG1 through promoter CpG island methylation has been reported in colorectal and cervical cancer but studies in breast cancer remain limited. METHODS: In silico analysis of human breast cancer patient data were used to demonstrate a correlation between DNA methylation and LRIG1 silencing in basal/triple-negative breast cancer, and its impact on patient survival. LRIG1 gene expression, protein abundance, and methylation enrichment were examined by quantitative reverse-transcription PCR, immunoblotting, and methylation immunoprecipitation, respectively, in breast cancer cell lines in vitro. We examined the impact of global demethylation on LRIG1 expression and methylation enrichment using 5-aza-2'-deoxycytidine. We also examined the effects of targeted demethylation of the LRIG1 CpG island, and transcriptional activation of LRIG1 expression, using the RNA guided deadCas9 transactivation system. RESULTS: Across breast cancer subtypes, LRIG1 expression is lowest in the basal/triple-negative subtype so we investigated whether differential methylation may contribute to this. Indeed, we find that LRIG1 CpG island methylation is most prominent in basal/triple-negative cell lines and patient samples. Use of the global demethylating agent 5-aza-2'-deoxycytidine decreases methylation leading to increased LRIG1 transcript expression in basal/triple-negative cell lines, while having no effect on LRIG1 expression in luminal/ER-positive cell lines. Using a CRISPR/deadCas9 (dCas9)-based targeting approach, we demonstrate that TET1-mediated demethylation (Tet1-dCas9) along with VP64-mediated transcriptional activation (VP64-dCas9) at the CpG island, increased endogenous LRIG1 expression in basal/triple-negative breast cancer cells, without transcriptional upregulation at predicted off-target sites. Activation of LRIG1 by the dCas9 transactivation system significantly increased LRIG1 protein abundance, reduced site-specific methylation, and reduced cancer cell viability. Our findings suggest that CRISPR-mediated targeted activation may be a feasible way to restore LRIG1 expression in cancer. CONCLUSIONS: Our study contributes novel insight into mechanisms which repress LRIG1 in triple-negative breast cancer and demonstrates for the first time that targeted de-repression of LRIG1 in cancer cells is possible. Understanding the epigenetic mechanisms associated with repression of tumour suppressor genes holds potential for the advancement of therapeutic approaches.

Regulation of the T-box transcription factor Tbx3 by the tumour suppressor microRNA-206 in 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.

Global analysis of ZNF217 chromatin occupancy in the breast cancer cell genome reveals an association with ERalpha.

BACKGROUND: The 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. RESULTS: ChIP-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. CONCLUSIONS: Our 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.

The Bacteriophages Therapy of Interdigital Pyoderma Complicated by Cellulitis with Antibiotic-Resistant Pseudomonas aeruginosa in a Dog-Case Report.

Pseudomonas aeruginosa is a highly pathogenic bacterium with high pathogenicity, that can cause serious infections in all species and especially in dogs. Treatment of the infection induced by this bacterium can be a challenge considering that some strains have developed resistance to most classes of antimicrobials. The use of bacteriophages to alleviate infections caused by Pseudomonas aeruginosa has demonstrated their potential for both internal and external applications. This study aimed to illustrate the treatment with bacteriophages in bacterially complicated skin lesions that do not respond to antimicrobial therapy.

VLDL lipolysis products increase VLDL fluidity and convert apolipoprotein E4 into a more expanded conformation.

Our previous work indicated that apolipoprotein (apo) E4 assumes a more expanded conformation in the postprandial period. The postprandial state is characterized by increased VLDL lipolysis. In this article, we tested the hypothesis that VLDL lipolysis products increase VLDL particle fluidity, which mediates expansion of apoE4 on the VLDL particle. Plasma from healthy subjects was collected before and after a moderately high-fat meal and incubated with nitroxyl-spin labeled apoE. ApoE conformation was examined by electron paramagnetic resonance spectroscopy using targeted spin probes on cysteines introduced in the N-terminal (S76C) and C-terminal (A241C) domains. Further, we synthesized a novel nitroxyl spin-labeled cholesterol analog, which gave insight into lipoprotein particle fluidity. Our data revealed that the order of lipoprotein fluidity was HDL approximately LDL

A Novel Bioengineered miR-127 Prodrug Suppresses the Growth and Metastatic Potential of Triple-Negative Breast Cancer Cells.

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-127PD), which we demonstrate is processed to mature, functional miR-127-3p in TNBC tumor cells. miR-127PD 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-127PD 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.

The LRIG family: enigmatic regulators of growth factor receptor signaling.

The leucine-rich repeats and immunoglobulin-like domains (LRIG) family of transmembrane proteins contains three vertebrate members (LRIG1, LRIG2 and 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 and LRIG3) and positively (Sma-10 and 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 that of 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.

Quantity control of the ErbB3 receptor tyrosine kinase at the endoplasmic reticulum.

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 steady-state levels of signaling-competent receptor are dictated by an ER-localized degradation pathway.

Lrig1 is an estrogen-regulated growth suppressor and correlates with longer relapse-free survival in ERα-positive breast cancer.

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 overexpressed 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 α (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 than in ERα-negative disease. Our study provides a molecular rationale for Lrig1 enrichment in ERα-positive disease by showing 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. In addition, 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 show 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.