Gabriel Eades

miR-200a Regulates SIRT1 Expression and Epithelial to Mesenchymal Transition (EMT)-like Transformation in Mammary Epithelial Cells

Evidence supports a critical role for microRNAs (miRNAs) in regulation of tissue-specific differentiation and development. Signifying a disruption of these programs, expression profiling has revealed extensive miRNA dysregulation in tumors compared with healthy tissue. The miR-200 family has been established as a key regulator of epithelial phenotype and, as such, is deeply involved in epithelial to mesenchymal transition (EMT) processes in breast cancer. However, the effects of the miR-200 family on transformation of normal mammary epithelial cells have yet to be fully characterized. By examining a TGF-β driven model of transformation of normal mammary epithelium, we demonstrate that the class III histone deacetylase silent information regulator 1 (SIRT1), a proposed oncogene in breast cancer, is overexpressed upon EMT-like transformation and that epigenetic silencing of miR-200a contributes at least in part to the overexpression of SIRT1. We have established the SIRT1 transcript as subject to regulation by miR-200a, through miR-200a targeting of SIRT1 3′-UTR. We also observed SIRT1 and miR-200a participation in a negative feedback regulatory loop. Restoration of miR-200a or the knockdown of SIRT1 prevented transformation of normal mammary epithelial cells evidenced by decreased anchorage-independent growth and decreased cell migration. Finally, we observed SIRT1 overexpression in association with decreased miR-200a in breast cancer patient samples. These observations provide further evidence for a critical tumor suppressive role of the miR-200 family in breast epithelium in addition to identifying a novel regulatory mechanism, which may contribute to SIRT1 up-regulation in breast cancer.

miR-200a regulates Nrf2 activation by targeting Keap1 mRNA in breast cancer cells.

NF-E2-related factor 2 (Nrf2) is an important transcription factor that activates the expression of cellular detoxifying enzymes. Nrf2 expression is largely regulated through the association of Nrf2 with Kelch-like ECH-associated protein 1 (Keap1), which results in cytoplasmic Nrf2 degradation. Conversely, little is known concerning the regulation of Keap1 expression. Until now, a regulatory role for microRNAs (miRs) in controlling Keap1 gene expression had not been characterized. By using miR array-based screening, we observed miR-200a silencing in breast cancer cells and demonstrated that upon re-expression, miR-200a targets the Keap1 3′-untranslated region (3′-UTR), leading to Keap1 mRNA degradation. Loss of this regulatory mechanism may contribute to the dysregulation of Nrf2 activity in breast cancer. Previously, we have identified epigenetic repression of miR-200a in breast cancer cells. Here, we find that treatment with epigenetic therapy, the histone deacetylase inhibitor suberoylanilide hydroxamic acid, restored miR-200a expression and reduced Keap1 levels. This reduction in Keap1 levels corresponded with Nrf2 nuclear translocation and activation of Nrf2-dependent NAD(P)H-quinone oxidoreductase 1 (NQO1) gene transcription. Moreover, we found that Nrf2 activation inhibited the anchorage-independent growth of breast cancer cells. Finally, our in vitro observations were confirmed in a model of carcinogen-induced mammary hyperplasia in vivo. In conclusion, our study demonstrates that miR-200a regulates the Keap1/Nrf2 pathway in mammary epithelium, and we find that epigenetic therapy can restore miR-200a regulation of Keap1 expression, therefore reactivating the Nrf2-dependent antioxidant pathway in breast cancer.

MiR-28 regulates Nrf2 expression through a Keap1-independent mechanism

NF-E2-related factor 2 (Nrf2) is an important transcription factor involved in antioxidant response. Nrf2 binds antioxidant response elements (ARE) within promoters of genes encoding detoxification enzymes (e.g., NAD (P) H-quinone oxidoreductase 1 (NQO1)) leading to their transcriptional activation. Nrf2 function is regulated post-translationally by its negative regulator Kelch-like ECH-associated protein 1 (Keap1) that binds Nrf2 and induces cytoplasmic Nrf2 degradation. Our present studies provide new evidence that Nrf2 expression can be regulated by a Keap1-independent mechanism. Here, we utilized breast epithelial cells to explore the impact of microRNA (miRNA) on Nrf2 expression. We found that Nrf2 mRNA levels are reversibly correlated with miR-28 expression and that ectopic expression of miR-28 alone reduces Nrf2 mRNA and protein levels. We further investigated the molecular mechanisms by which miR-28 inhibits Nrf2 mRNA expression. Initially, the ability of miR-28 to regulate the 3′ untranslated region (3′UTR) of Nrf2 mRNA was evaluated via luciferase reporter assay. We observed that miR-28 reduces wild-type Nrf2 3′UTR luciferase reporter activity and this repression is eliminated upon mutation of the miR-28 targeting seed sequence within the Nrf2 3′UTR. Moreover, over-expression of miR-28 decreased endogenous Nrf2 mRNA and protein expression. We also explored the impact of miR-28 on Keap1-Nrf2 interactions and found that miR-28 over-expression does not alter Keap1 protein levels and has no effect on the interaction of Keap1 and Nrf2. Our findings, that miR-28 targets the 3′UTR of Nrf2 mRNA and decreases Nrf2 expression, suggest that this miRNA is involved in the regulation of Nrf2 expression in breast epithelial cells.

lincRNA-RoR and miR-145 Regulate Invasion in Triple-Negative Breast Cancer via Targeting ARF6

Triple-negative (ER−, HER2−, PR−) breast cancer (TNBC) is an aggressive disease with a poor prognosis with no available molecularly targeted therapy. Silencing of microRNA-145 (miR-145) may be a defining marker of TNBC based on molecular profiling and deep sequencing. Therefore, the molecular mechanism behind miR-145 downregulation in TNBC was examined. Overexpression of the long intergenic noncoding RNA regulator of reprogramming, lincRNA-RoR, functions as a competitive endogenous RNA sponge in TNBC. Interestingly, lincRNA-RoR is dramatically upregulated in TNBC and in metastatic disease and knockdown restores miR-145 expression. Previous reports suggest that miR-145 has growth-suppressive activity in some breast cancers; however, these data in TNBC indicate that miR-145 does not affect proliferation or apoptosis but instead, miR-145 regulates tumor cell invasion. Investigation of miR-145-regulated pathways involved in tumor invasion revealed a novel target, the small GTPase ADP-ribosylation factor 6 (Arf6). Subsequent analysis demonstrated that ARF6, a known regulator of breast tumor cell invasion, is dramatically upregulated in TNBC and in breast tumor metastasis. Mechanistically, ARF6 regulates E-cadherin localization and affects cell–cell adhesion. These results reveal a lincRNA-RoR/miR-145/ARF6 pathway that regulates invasion in TNBCs. Implications: The lincRNA-RoR/miR-145/ARF6 pathway is critical to TNBC metastasis and could serve as biomarkers or therapeutic targets for improving survival. Mol Cancer Res; 13(2); 330–8. ©2014 AACR.

Estrogen Receptor α Signaling Regulates Breast Tumor-initiating Cells by Down-regulating miR-140 Which Targets the Transcription Factor SOX2

Background: miR-140 is down-regulated in non-invasive and invasive breast tumors compared with normal breast tissues. Results: Estrogen receptor α signaling down-regulates miR-140 in breast cancer where miR-140 targets embryonic transcription factor SOX2. Conclusion: ERα signaling regulates breast tumor-initiating cells through modulating miR-140 targeting of SOX2. Significance: Understanding cancer stem cell biology may reveal biomarkers or targets for therapeutic intervention. Several reports have indicated that miR-140, a possible tumor suppressor microRNA (miR), is down-regulated in breast tumors compared with normal breast tissues. However, the role of miR-140 in breast tumorigenesis is unclear. We initiated studies that examined estrogen receptor α (ERα) signaling in the tissue-specific regulation of miR-140 in breast cancer. We found that estrogen stimulation of ERα-positive breast cancer cells resulted in decreased miR-140 expression. We performed promoter analyses and examined predicted ERα binding elements in the miR-140 promoter using luciferase constructs of a miR-140 promoter deletion series. Our studies revealed that ERα binds to one specific estrogen response element flanking the miR-140 promoter and consequently suppresses miR-140 transcription. We found that the stem cell self-renewal regulator SOX2 is a novel target of miR-140, and that this miR-140/SOX2 pathway critically regulates breast tumor-initiating cell survival, providing a new link between ERα signaling and breast cancer stem cell maintenance.

Characterization of a stem-like subpopulation in basal-like ductal carcinoma in situ (DCIS) lesions.

Background: Very little is known about cancer stem cells in ductal carcinoma in situ (DCIS). Results: Basal-like DCIS contain CD49f+/CD24− ALDH1+ stem-like subpopulations with enhanced migratory capacity, unique exosomal microRNA signature, and sulforaphane sensitivity. Conclusion: We characterized basal-like DCIS stem cells and demonstrate a potential chemopreventive strategy. Significance: DCIS stem cells are important therapeutic targets for preventing recurrence or progression to invasive disease. Previously, we found that basal-like ductal carcinoma in situ (DCIS) contains cancer stem-like cells. Here, we characterize stem-like subpopulations in a model of basal-like DCIS and identify subpopulations of CD49f+/CD24− stem-like cells that possess aldehyde dehydrogenase 1 activity. We found that these cells show enhanced migration potential compared with non-stem DCIS cells. We also found that the chemopreventive agent sulforaphane can target these DCIS stem-like cells, reduce aldehyde dehydrogenase 1 (ALDH1) expression, and decrease mammosphere and progenitor colony formation. Furthermore, we characterized exosomal trafficking of microRNAs in DCIS and found that several microRNAs (miRs) including miR-140, miR-29a, and miR-21 are differentially expressed in exosomes from DCIS stem-like cells. We found that SFN treatment could reprogram DCIS stem-like cells as evidenced by significant changes in exosomal secretion more closely resembling that of non-stem cancer cells. Finally, we demonstrated that exosomal secretion of miR-140 might impact signaling in nearby breast cancer cells.

Long non-coding RNAs in stem cells and cancer

An overwhelming majority of the transcribed genome encodes for non-coding RNA (ncRNA) sequences. Deep sequencing of the transcriptome has uncovered tens of thousands of long ncRNA (lncRNA) sequences. However, little is known regarding the possible functions for a vast majority of these sequences. Among those lncRNAs whose function has been experimentally validated, most serve as regulators of gene expression. LncRNAs have been found to be critical to development and homeostasis and they have been implicated in several pathologies including cancer. Here, we examine the functions and underlying mechanisms of lncRNAs in stem cells and in cancer biology, areas linked by the actions of lncRNAs.

Targeting exosomes from preadipocytes inhibits preadipocyte to cancer stem cell signaling in early-stage breast cancer.

The tumor microenvironment plays a critical role in regulating breast tumor progression. Signaling between preadipocytes and breast cancer cells has been found to promote breast tumor formation and metastasis. Exosomes secreted from preadipocytes are important components of the cancer stem cell niche. Mouse preadipocytes (3T3L1) are treated with the natural antitumor compound shikonin (SK) and exosomes derived from mouse preadipocytes are co-cultured with MCF10DCIS cells. We examine how preadipocyte-derived exosomes can regulate early-stage breast cancer via regulating stem cell renewal, cell migration, and tumor formation. We identify a critical miR-140/SOX2/SOX9 axis that regulates differentiation, stemness, and migration in the tumor microenvironment. Next, we find that the natural antitumor compound SK can inhibit preadipocyte signaling inhibiting nearby ductal carcinoma in situ (DCIS) cells. Through co-culture experiments, we find that SK-treated preadipocytes secrete exosomes with high levels of miR-140, which can impact nearby DCIS cells through targeting SOX9 signaling. Finally, we find that preadipocyte-derived exosomes promote tumorigenesis in vivo, providing strong support for the importance of exosomal signaling in the tumor microenvironment. Our data also show that targeting the tumor microenvironment may assist in blocking tumor progression.

The Krüppel-like factor 2 and Krüppel-like factor 4 genes interact to maintain endothelial integrity in mouse embryonic vasculogenesis

BackgroundKrüppel-like Factor 2 (KLF2) plays an important role in vessel maturation during embryonic development. In adult mice, KLF2 regulates expression of the tight junction protein occludin, which may allow KLF2 to maintain vascular integrity. Adult tamoxifen-inducible Krüppel-like Factor 4 (KLF4) knockout mice have thickened arterial intima following vascular injury. The role of KLF4, and the possible overlapping functions of KLF2 and KLF4, in the developing vasculature are not well-studied.ResultsEndothelial breaks are observed in a major vessel, the primary head vein (PHV), in KLF2-/-KLF4-/- embryos at E9.5. KLF2-/-KLF4-/- embryos die by E10.5, which is earlier than either single knockout. Gross hemorrhaging of multiple vessels may be the cause of death. E9.5 KLF2-/-KLF4+/- embryos do not exhibit gross hemorrhaging, but cross-sections display disruptions of the endothelial cell layer of the PHV, and these embryos generally also die by E10.5. Electron micrographs confirm that there are gaps in the PHV endothelial layer in E9.5 KLF2-/-KLF4-/- embryos, and show that the endothelial cells are abnormally bulbous compared to KLF2-/- and wild-type (WT). The amount of endothelial Nitric Oxide Synthase (eNOS) mRNA, which encodes an endothelial regulator, is reduced by 10-fold in E9.5 KLF2-/-KLF4-/- compared to KLF2-/- and WT embryos. VEGFR2, an eNOS inducer, and occludin, a tight junction protein, gene expression are also reduced in E9.5 KLF2-/-KLF4-/- compared to KLF2-/- and WT embryos.ConclusionsThis study begins to define the roles of KLF2 and KLF4 in the embryonic development of blood vessels. It indicates that the two genes interact to maintain an intact endothelial layer. KLF2 and KLF4 positively regulate the eNOS, VEGFR2 and occludin genes. Down-regulation of these genes in KLF2-/-KLF4-/- embryos may result in the observed loss of vascular integrity.

Roles of microRNA-140 in stem cell-associated early stage breast cancer.

An increasing body of evidence supports a stepwise model for progression of breast cancer from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC). Due to the high level of DCIS heterogeneity, we cannot currently predict which patients are at highest risk for disease recurrence or progression. The mechanisms of progression are still largely unknown, however cancer stem cell populations in DCIS lesions may serve as malignant precursor cells intimately involved in progression. While genetic and epigenetic alterations found in DCIS are often shared by IDC, mRNA and miRNA expression profiles are significantly altered. Therapeutic targeting of cancer stem cell pathways and differentially expressed miRNA could have significant clinical benefit. As tumor grade increases, miRNA-140 is progressively downregulated. miR-140 plays an important tumor suppressive role in the Wnt, SOX2 and SOX9 stem cell regulator pathways. Downregulation of miR-140 removes inhibition of these pathways, leading to higher cancer stem cell populations and breast cancer progression. miR-140 downregulation is mediated through both an estrogen response element in the miR-140 promoter region and differential methylation of CpG islands. These mechanisms are novel targets for epigenetic therapy to activate tumor suppressor signaling via miR-140. Additionally, we briefly explored the emerging role of exosomes in mediating intercellular miR-140 signaling. The purpose of this review is to examine the cancer stem cell signaling pathways involved in breast cancer progression, and the role of dysregulation of miR-140 in regulating DCIS to IDC transition.