Benjamin Wolfson

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.

MicroRNA 140 Promotes Expression of Long Noncoding RNA NEAT1 in Adipogenesis

ABSTRACT More than 40% of the U.S. population are clinically obese and suffer from metabolic syndrome with an increased risk of postmenopausal estrogen receptor-positive breast cancer. Adipocytes are the primary component of adipose tissue and are formed through adipogenesis from precursor mesenchymal stem cells. While the major molecular pathways of adipogenesis are understood, little is known about the noncoding RNA signaling networks involved in adipogenesis. Using adipocyte-derived stem cells (ADSCs) isolated from wild-type and microRNA 140 (miR-140) knockout mice, we identify a novel miR-140/long noncoding RNA (lncRNA) NEAT1 signaling network necessary for adipogenesis. miR-140 knockout ADSCs have dramatically decreased adipogenic capabilities associated with downregulation of NEAT1 expression. We identified a miR-140 binding site in NEAT1 and found that mature miR-140 in the nucleus can physically interact with NEAT1, leading to increased NEAT1 expression. We demonstrated that reexpression of NEAT1 in miR-140 knockout ADSCs is sufficient to restore their ability to undergo differentiation. Our results reveal an exciting new noncoding RNA signaling network that regulates adipogenesis and that is a potential new target in the prevention or treatment of obesity.

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.

Dysregulation of the BRCA1/long non-coding RNA NEAT1 signaling axis contributes to breast tumorigenesis.

Dysregulation of long non-codng RNA (lncRNA) expression has been found to contribute to tumorigenesis. However, the roles of lncRNAs in BRCA1-related breast cancer remain largely unknown. In this study, we delineate the role of the novel BRCA1/lncRNA NEAT1 signaling axis in breast tumorigenesis. BRCA1 inhibits NEAT1 expression potentially through binding to its genomic binding site upstream of the NEAT1 gene. BRCA1 deficiency in human normal/cancerous breast cells and mouse mammary glands leads to NEAT1 overexpression. Our studies show that NEAT1 upregulation resulting from BRCA1 deficiency stimulates in vitro and in vivo breast tumorigenicity. We have further identified molecular mediators downstream of the BRCA1/NEAT1 axis. NEAT1 epigenetically silences miR-129-5p expression by promoting the DNA methylation of the CpG island in the miR-129 gene. Silencing of miR-129-5p expression by NEAT1 results in upregulation of WNT4 expression, a target of miR-129-5p, which leads to activation of oncogenic WNT signaling. Our functional studies indicate that this NEAT1/miR-129-5p/WNT4 axis contributes to the tumorigenic effects of BRCA1 deficiency. Finally our in silico expression correlation analysis suggests the existence of the BRCA1/NEAT1/miR-129-5p axis in breast cancer. Our findings, taken together, suggest that the dysregulation of the BRCA1/NEAT1/miR-129-5p/WNT4 signaling axis is involved in promoting breast tumorigenesis.

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.

Adipocyte activation of cancer stem cell signaling in breast cancer

Signaling within the tumor microenvironment has a critical role in cancer initiation and progression. Adipocytes, one of the major components of the breast microenvironment, have been shown to provide pro-tumorigenic signals that promote cancer cell proliferation and invasiveness in vitro and tumorigenicity in vivo. Adipocyte secreted factors such as leptin and interleukin-6 (IL-6) have a paracrine effect on breast cancer cells. In adipocyte-adjacent breast cancer cells, the leptin and IL-6 signaling pathways activate janus kinase 2/signal transducer and activator of transcription 5, promoting the epithelial-mesenchymal transition, and upregulating stemness regulators such as Notch, Wnt and the Sex determining region Y-box 2/octamer binding transcription factor 4/Nanog signaling axis. In this review we will summarize the major signaling pathways that regulate cancer stem cells in breast cancer and describe the effects that adipocyte secreted IL-6 and leptin have on breast cancer stem cell signaling. Finally we will introduce a new potential treatment paradigm of inhibiting the adipocyte-breast cancer cell signaling via targeting the IL-6 or leptin pathways.

NRF2/miR-140 signaling confers radioprotection to human lung fibroblasts

Breast and lung cancer patients who are treated with radiotherapy often have severe side effects, including radiation-induced lung damage and secondary cancers. Activation of the NRF2 pathway is a well-known mechanism that protects cells against radiation induced oxidative stress, but its role in radiation-induced lung damage is not well understood. Using human lung fibroblasts (HLFs) we found that ionizing radiation (IR) leads to BRCA1-dependent activation of NRF2 through the inhibition of KEAP1 function, promoting the nuclear accumulation of NRF2, and activating critical radioprotective mechanisms. We discovered that NRF2 directly binds to the miR-140 promoter and increases its expression in response to IR treatment. Gain and loss of function studies further showed the ability of miR-140 to regulate lung fibroblast self-renewal upon irradiation, a potential mechanism to contribute to the regulation of DNA repair. We verified our in vitro findings using primary lung fibroblast cultures from wild type and Nrf2 (KO) mice. Using these models we showed that IR induces overexpression of Brca1, Nrf2 and miR-140 in lung tissue after irradiation. These data reveal a novel radioprotective mechanism in which IR promotes NRF2 nuclear translocation and subsequent activation of miR-140 transcription in HLFs.

Noncoding RNAs in breast cancer

The mammalian transcriptome has recently been revealed to encompass a large number of noncoding RNAs (ncRNAs) that play a variety of important regulatory roles in gene expression and other biological processes. MicroRNAs (miRNAs), the best studied of the short noncoding RNAs (sncRNAs), have been extensively characterized with regard to their biogenesis, function and importance in tumorigenesis. Another class of sncRNAs called piwi-interacting RNAs (piRNAs) has also gained attention recently in cancer research owing to their critical role in stem cell regulation. Long noncoding RNAs (lncRNAs) of >200 nucleotides in length have recently emerged as key regulators of developmental processes, including mammary gland development. lncRNA dysregulation has also been implicated in the development of various cancers, including breast cancer. In this review, we describe and discuss the roles of sncRNAs (including miRNAs and piRNAs) and lncRNAs in the initiation and progression of breast tumorigenesis, with a focus on outlining the molecular mechanisms of oncogenic and tumor-suppressor ncRNAs. Moreover, the current and potential future applications of ncRNAs to clinical breast cancer research are also discussed, with an emphasis on ncRNA-based diagnosis, prognosis and future therapeutics.

Mechanisms of the alternative activation of macrophages and non-coding RNAs in the development of radiation-induced lung fibrosis

Radiation-induced lung fibrosis (RILF) is a common side effect of thoracic irradiation therapy and leads to high mortality rates after cancer treatment. Radiation injury induces inflammatory M1 macrophage polarization leading to radiation pneumonitis, the first stage of RILF progression. Fibrosis occurs due to the transition of M1 macrophages to the anti-inflammatory pro-fibrotic M2 phenotype, and the resulting imbalance of macrophage regulated inflammatory signaling. Non-coding RNA signaling has been shown to play a large role in the regulation of the M2 mediated signaling pathways that are associated with the development and progression of fibrosis. While many studies show the link between M2 macrophages and fibrosis, there are only a few that explore their distinct role and the regulation of their signaling by non-coding RNA in RILF. In this review we summarize the current body of knowledge describing the roles of M2 macrophages in RILF, with an emphasis on the expression and functions of non-coding RNAs.

High-fat diet promotes mammary gland myofibroblast differentiation through miR-140 downregulation

ABSTRACT Human breast adipose tissue is a heterogeneous cell population consisting of mature white adipocytes, multipotent mesenchymal stem cells, committed progenitor cells, fibroblasts, endothelial cells, and immune cells. Dependent on external stimulation, adipose-derived stem cells differentiate along diverse lineages into adipocytes, chondrocytes, osteoblasts, fibroblasts, and myofibroblasts. It is currently not fully understood how a high-fat diet reprograms adipose-derived stem cells into myofibroblasts. In our study, we used mouse models of a regular diet and of high-fat-diet-induced obesity to investigate the role of dietary fat on myofibroblast differentiation in the mammary stromal microenvironment. We found that a high-fat diet promotes myofibroblast differentiation by decreasing microRNA 140 (miR-140) expression in mammary adipose tissue through a novel negative-feedback loop. Increased transforming growth factor β1 (TGF-β1) in mammary adipose tissue in obese mice activates SMAD3 signaling, causing phospho-SMAD3 to bind to the miR-140 locus and inhibit miR-140 transcription. This prevents miR-140 from targeting SMAD3 for degradation, resulting in amplified TGF-β1/SMAD3 signaling and miR-140 downregulation-dependent myofibroblast differentiation. Using tissue and coculture models, we found that myofibroblasts and the fibrotic microenvironment created by myofibroblasts impact the stemness and proliferation of normal ductal epithelial cells and early-stage breast cancer invasion and stemness.