Reciprocal Signaling Research Articles

Proteome reprogramming of endometrial epithelial cells by human trophectodermal small extracellular vesicles reveals key insights into embryo implantation.

Embryo implantation into the receptive endometrium is critical in pregnancy establishment, initially requiring reciprocal signalling between outer layer of the blastocyst (trophectoderm cells) and endometrial epithelium; however, factors regulating this crosstalk remain poorly understood. Although endometrial extracellular vesicles (EVs) are known to signal to the embryo during implantation, the role of embryo-derived EVs remains largely unknown. Here, we provide a comprehensive proteomic characterisation of a major class of EVs, termed small EVs (sEVs), released by human trophectoderm cells (Tsc-sEVs) and their capacity to reprogram protein landscape of endometrial epithelium in vitro. Highly purified Tsc-sEVs (30-200 nm, ALIX+ , TSG101+ , CD9/63/81+ ) were enriched in known players of implantation (LIFR, ICAM1, TAGLN2, WNT5A, FZD7, ROR2, PRICKLE2), antioxidant activity (SOD1, PRDX1/4/6), tissue integrity (EZR, RAC1, RHOA, TNC), and focal adhesions (FAK, ITGA2/V, ITGB1/3). Functionally, Tsc-sEVs were taken up by endometrial cells, altered transepithelial electrical resistance, and upregulated proteins implicated in embryo attachment (ITGA2/V, ITGB1/3), immune regulation (CD59, CD276, LGALS3), and antioxidant activity (GPX1/3/4, PRDX1/2/4/5/6): processes that are critical for successful implantation. Collectively, we provide critical insights into Tsc-sEV-mediated regulation of endometrial function that contributes to our understanding of the molecular basis of implantation.

Pretreatment of aged mice with retinoic acid supports alveolar regeneration via upregulation of reciprocal PDGFA signalling

ObjectivesIdiopathic pulmonary fibrosis (IPF) primarily affects the aged population and is characterised by failure of alveolar regeneration, leading to loss of alveolar type 1 (AT1) cells. Aged mouse models of...

Re-Evaluating the Oxidative Phenotype: Can Endurance Exercise Save the Western World?

Mitochondria are popularly called the “powerhouses” of the cell. They promote energy metabolism through the tricarboxylic acid (TCA) cycle and oxidative phosphorylation, which in contrast to cytosolic glycolysis are oxygen-dependent and significantly more substrate efficient. That is, mitochondrial metabolism provides substantially more cellular energy currency (ATP) per macronutrient metabolised. Enhancement of mitochondrial density and metabolism are associated with endurance training, which allows for the attainment of high relative VO2 max values. However, the sedentary lifestyle and diet currently predominant in the Western world lead to mitochondrial dysfunction. Underdeveloped mitochondrial metabolism leads to nutrient-induced reducing pressure caused by energy surplus, as reduced nicotinamide adenine dinucleotide (NADH)-mediated high electron flow at rest leads to “electron leak” and a chronic generation of superoxide radicals (O2−). Chronic overload of these reactive oxygen species (ROS) damages cell components such as DNA, cell membranes, and proteins. Counterintuitively, transiently generated ROS during exercise contributes to adaptive reduction-oxidation (REDOX) signalling through the process of cellular hormesis or “oxidative eustress” defined by Helmut Sies. However, the unaccustomed, chronic oxidative stress is central to the leading causes of mortality in the 21st century—metabolic syndrome and the associated cardiovascular comorbidities. The endurance exercise training that improves mitochondrial capacity and the protective antioxidant cellular system emerges as a universal intervention for mitochondrial dysfunction and resultant comorbidities. Furthermore, exercise might also be a solution to prevent ageing-related degenerative diseases, which are caused by impaired mitochondrial recycling. This review aims to break down the metabolic components of exercise and how they translate to athletic versus metabolically diseased phenotypes. We outline a reciprocal relationship between oxidative metabolism and inflammation, as well as hypoxia. We highlight the importance of oxidative stress for metabolic and antioxidant adaptation. We discuss the relevance of lactate as an indicator of critical exercise intensity, and inferring from its relationship with hypoxia, we suggest the most appropriate mode of exercise for the case of a lost oxidative identity in metabolically inflexible patients. Finally, we propose a reciprocal signalling model that establishes a healthy balance between the glycolytic/proliferative and oxidative/prolonged-ageing phenotypes. This model is malleable to adaptation with oxidative stress in exercise but is also susceptible to maladaptation associated with chronic oxidative stress in disease. Furthermore, mutations of components involved in the transcriptional regulatory mechanisms of mitochondrial metabolism may lead to the development of a cancerous phenotype, which progressively presents as one of the main causes of death, alongside the metabolic syndrome.

Genetic and Cellular Interaction During Cardiovascular Development Implicated in Congenital Heart Diseases.

Congenital heart disease (CHD) is the most common life-threatening congenital anomaly. CHD occurs due to defects in cardiovascular development, and the majority of CHDs are caused by a multifactorial inheritance mechanism, which refers to the interaction between genetic and environmental factors. During embryogenesis, the cardiovascular system is derived from at least four distinct cell lineages: the first heart field, second heart field, cardiac neural crest, and proepicardial organ. Understanding the genes involved in each lineage is essential to uncover the genomic architecture of CHD. Therefore, we provide an overview of recent research progress using animal models and mutation analyses to better understand the molecular mechanisms and pathways linking cardiovascular development and CHD. For example, we highlight our recent work on genes encoding three isoforms of inositol 1,4,5-trisphosphate receptors (IP3R1, 2, and 3) that regulate various vital and developmental processes, which have genetic redundancy during cardiovascular development. Specifically, IP3R1 and 2 have redundant roles in the atrioventricular cushion derived from the first heart field lineage, whereas IP3R1 and 3 exhibit redundancy in the right ventricle and the outflow tract derived from the second heart field lineage, respectively. Moreover, 22q11.2 deletion syndrome (22q11DS) is highly associated with CHD involving the outflow tract, characterized by defects of the cardiac neural crest lineage. However, our studies have shown that TBX1, a major genetic determinant of 22q11DS, was not expressed in the cardiac neural crest but rather in the second heart field, suggesting the importance of the cellular interaction between the cardiac neural crest and the second heart field. Comprehensive genetic analysis using the Japanese genome bank of CHD and mouse models revealed that a molecular regulatory network involving GATA6, FOXC1/2, TBX1, SEMA3C, and FGF8 was essential for reciprocal signaling between the cardiac neural crest and the second heart field during cardiovascular development. Elucidation of the genomic architecture of CHD using induced pluripotent stem cells and next-generation sequencing technology, in addition to genetically modified animal models and human mutation analyses, would facilitate the development of regenerative medicine and/or preventive medicine for CHD in the near future.

Neuron-Oligodendrocyte Interactions in the Structure and Integrity of Axons.

The myelination of axons by oligodendrocytes is a highly complex cell-to-cell interaction. Oligodendrocytes and axons have a reciprocal signaling relationship in which oligodendrocytes receive cues from axons that direct their myelination, and oligodendrocytes subsequently shape axonal structure and conduction. Oligodendrocytes are necessary for the maturation of excitatory domains on the axon including nodes of Ranvier, help buffer potassium, and support neuronal energy metabolism. Disruption of the oligodendrocyte-axon unit in traumatic injuries, Alzheimer’s disease and demyelinating diseases such as multiple sclerosis results in axonal dysfunction and can culminate in neurodegeneration. In this review, we discuss the mechanisms by which demyelination and loss of oligodendrocytes compromise axons. We highlight the intra-axonal cascades initiated by demyelination that can result in irreversible axonal damage. Both the restoration of oligodendrocyte myelination or neuroprotective therapies targeting these intra-axonal cascades are likely to have therapeutic potential in disorders in which oligodendrocyte support of axons is disrupted.

SERS-Microfluidic Approach for the Quantitative Detection of miRNA Using DNAzyme-Mediated Reciprocal Signal Amplification.

MicroRNAs (miRNAs) play important roles in biological processes. Designing a sensitive, selective, and rapid method of miRNA detection is crucial for biological research. Here, with a reciprocal signal amplification (RSA) probe, this work established a novel surface-enhanced Raman scattering (SERS)-microfluidic approach for the quantitative analysis of miRNA. First, via a DNAzyme self-assemble cycle reaction, two types of SERS signals produce amplified reciprocal changes. The sum of the absolute signal value is first adopted for the quantitative analysis of miRNA, which results in an enhanced response and a reduced blank value. Furthermore, the assay is integrated in an electric drive microfluidic mixing reactor that enables physical mixing and enriching of the reactants for more rapid and enhanced detection sensitivity. The protocol owns the merits of the SERS technology, amplified reciprocal signals, and a microfluidic chip, with a detection limit of 2.92 fM for miR-141 in 40 min. In addition, successful determination of miRNA in a variety of cells proved the practicability of the assay. Compared with the reported strategies for miRNA analysis, this work avoids a complex and time-consuming procedure and enhances the sensitivity and specificity. The method opens a promising way for biomolecular chip detection and research.

Fully defined human pluripotent stem cell-derived microglia and tri-culture system model C3 production in Alzheimer's disease.

Aberrant inflammation in the central nervous system (CNS) has been implicated as a major player in the pathogenesis of human neurodegenerative disease. We developed a novel approach to derive microglia from human pluripotent stem cells (hPSCs) and built a defined hPSC-derived tri-culture system containing pure populations of hPSC-derived microglia, astrocytes, and neurons to dissect cellular crosstalk along the neuroinflammatory axis in vitro.We used the tri-culture system to model neuroinflammation in Alzheimer’s Disease with hPSCs harboring the APPSWE+/+ mutation and their isogenic control. We found that complement C3, a protein that is increased under inflammatory conditions and implicated in synaptic loss, is potentiated in tri-culture and further enhanced in APPSWE+/+ tri-cultures due to microglia initiating reciprocal signaling with astrocytes to produce excess C3. Our study defines the major cellular players contributing to increased C3 in AD and presents a broadly applicable platform to study neuroinflammation in human disease.

Gain-of-function p53 protein transferred via small extracellular vesicles promotes conversion of fibroblasts to a cancer-associated phenotype

SUMMARYTumor and stromal interactions consist of reciprocal signaling through cytokines, growth factors, direct cell-cell interactions, and extracellular vesicles (EVs). Small EVs (≤200 nm) have been considered critical messengers of cellular communication during tumor development. Here, we demonstrate that gain-of-function (GOF) p53 protein can be packaged into small EVs and transferred to fibroblasts. GOF p53 protein is selectively bound by heat shock protein 90 (HSP90), a chaperone protein, and packaged into small EVs. Inhibition of HSP90 activity blocks packaging of GOF, but not wild-type, p53 in small EVs. GOF p53-containing small EVs result in their conversion to cancer-associated fibroblasts. In vivo studies reveal that GOF p53-containing small EVs can enhance tumor growth and promote fibroblast transformation into a cancer-associated phenotype. These findings provide a better understanding of the complex interactions between cancer and stromal cells and may have therapeutic implications.

Reciprocal interaction between mesenchymal stem cells and transit amplifying cells regulates tissue homeostasis.

Interaction between adult stem cells and their progeny is critical for tissue homeostasis and regeneration. In multiple organs, mesenchymal stem cells (MSCs) give rise to transit amplifying cells (TACs), which then differentiate into different cell types. However, whether and how MSCs interact with TACs remains unknown. Using the adult mouse incisor as a model, we present in vivo evidence that TACs and MSCs have distinct genetic programs and engage in reciprocal signaling cross talk to maintain tissue homeostasis. Specifically, an IGF-WNT signaling cascade is involved in the feedforward from MSCs to TACs. TACs are regulated by tissue-autonomous canonical WNT signaling and can feedback to MSCs and regulate MSC maintenance via Wnt5a/Ror2-mediated non-canonical WNT signaling. Collectively, these findings highlight the importance of coordinated bidirectional signaling interaction between MSCs and TACs in instructing mesenchymal tissue homeostasis, and the mechanisms identified here have important implications for MSC-TAC interaction in other organs.

Anomalous incisor morphology indicates tissue-specific roles for Tfap2a and Tfap2b in tooth development

Mice possess two types of teeth that differ in their cusp patterns; incisors have one cusp and molars have multiple cusps. The patterning of these two types of teeth relies on fine-tuning of the reciprocal molecular signaling between dental epithelial and mesenchymal tissues during embryonic development. The AP-2 transcription factors, particularly Tfap2a and Tfap2b, are essential components of such epithelial-mesenchymal signaling interactions that coordinate craniofacial development in mice and other vertebrates, but little is known about their roles in the regulation of tooth development and shape. Here we demonstrate that incisors and molars differ in their temporal and spatial expression of Tfap2a and Tfap2b. At the bud stage, Tfap2a is expressed in both the epithelium and mesenchyme of the incisors and molars, but Tfap2b expression is restricted to the molar mesenchyme, only later appearing in the incisor epithelium. Tissue-specific deletions show that loss of the epithelial domain of Tfap2a and Tfap2b affects the number and spatial arrangement of the incisors, notably resulting in duplicated lower incisors. In contrast, deletion of these two genes in the mesenchymal domain has little effect on tooth development. Collectively these results implicate epithelial expression of Tfap2a and Tfap2b in regulating the extent of the dental lamina associated with patterning the incisors and suggest that these genes contribute to morphological differences between anterior (incisor) and posterior (molar) teeth within the mammalian dentition.

Dysregulation of intercellular signaling by MOF deletion leads to liver injury

Epigenetic mechanisms that alter heritable gene expression and chromatin structure play an essential role in many biological processes, including liver function. Human MOF (males absent on the first) is a histone acetyltransferase that is globally downregulated in human steatohepatitis. However, the function of MOF in the liver remains unclear. Here, we report that MOF plays an essential role in adult liver. Genetic deletion of Mof by Mx1-Cre in the liver leads to acute liver injury, with increase of lipid deposition and fibrosis akin to human steatohepatitis. Surprisingly, hepatocyte-specific Mof deletion had no overt liver abnormality. Using the in vitro coculturing experiment, we show that Mof deletion-induced liver injury requires coordinated changes and reciprocal signaling between hepatocytes and Kupffer cells, which enables feedforward regulation to augment inflammation and apoptotic responses. At the molecular level, Mof deletion induced characteristic changes in metabolic gene programs, which bore noticeable similarity to the molecular signature of human steatohepatitis. Simultaneous deletion of Mof in both hepatocytes and macrophages results in enhanced expression of inflammatory genes and NO signaling in vitro. These changes, in turn, lead to apoptosis of hepatocytes and lipotoxicity. Our work highlights the importance of histone acetyltransferase MOF in maintaining metabolic liver homeostasis and sheds light on the epigenetic dysregulation in liver pathogenesis.

Form and function of the apical extracellular matrix: new insights from Caenorhabditis elegans, Drosophila melanogaster, and the vertebrate inner ear.

Apical extracellular matrices (aECMs) are the extracellular layers on the apical sides of epithelia. aECMs form the outer layer of the skin in most animals and line the luminal surface of internal tubular epithelia. Compared to the more conserved basal ECMs (basement membranes), aECMs are highly diverse between tissues and between organisms and have been more challenging to understand at mechanistic levels. Studies in several genetic model organisms are revealing new insights into aECM composition, biogenesis, and function and have begun to illuminate common principles and themes of aECM organization. There is emerging evidence that, in addition to mechanical or structural roles, aECMs can participate in reciprocal signaling with associated epithelia and other cell types. Studies are also revealing mechanisms underlying the intricate nanopatterns exhibited by many aECMs. In this review, we highlight recent findings from well-studied model systems, including the external cuticle and ductal aECMs of Caenorhabditis elegans, Drosophila melanogaster, and other insects and the internal aECMs of the vertebrate inner ear.

An RGDKGE-Containing Cryptic Collagen Fragment Regulates Phosphorylation of Large Tumor Suppressor Kinase-1 and Controls Ovarian Tumor Growth by a Yes-Associated Protein–Dependent Mechanism

The growth and spread of malignant tumors, such as ovarian carcinomas, are governed in part by complex interconnected signaling cascades occurring between stromal and tumor cells. These reciprocal cross-talk signaling networks operating within the local tissue microenvironment may enhance malignant tumor progression. Understanding how novel bioactive molecules generated within the tumor microenvironment regulate signaling pathways in distinct cellular compartments is critical for the development of more effective treatment paradigms. Herein, we provide evidence that blocking cellular interactions with an RGDKGE-containing collagen peptide that selectively binds integrin β3 on ovarian tumor cells enhances the phosphorylation of the hippo effector kinase large tumor suppressor kinase-1 and reduces nuclear accumulation of yes-associated protein and its target gene c-Myc. Selectively targeting this RGDKGE-containing collagen fragment inhibited ovarian tumor growth and the development of ascites fluid invivo. These findings suggest that this bioactive collagen fragment may represent a previously unknown regulator of the hippo effector kinase large tumor suppressor kinase-1 and regulate ovarian tumor growth by a yes-associated protein-dependent mechanism. Taken together, these data not only provide new mechanistic insight into how a unique collagen fragment may regulate ovarian cancer, but in addition may help provide a useful new alternative strategy to control ovarian tumor progression based on selectively disrupting a previously unappreciated signaling cascade.

SNAIL Transctiption factor in prostate cancer cells promotes neurite outgrowth

Neurite outgrowth involves reciprocal signaling interactions between tumor cells and nerves where invading tumor cells have acquired the ability to respond to pro-invasive signals within the nerve environment. Neurite outgrowth could serve as a mechanism leading to invasion of cancer cells into the nerve sheath and subsequent metastasis. Snail transcription factor can promote migration and invasion of prostate cancer cells. We hypothesized that prostate cancer cell interaction with nerve cells will be mediated by Snail expression within prostate cancer cells. For this study we utilized various prostate cancer cell lines: C4-2 non-silencing (NS, control); C4-2 Snail shRNA, (stable Snail knockdown); LNCaP Neo (empty vector control) and LNCaP Snail (stably over-expressing Snail). Cancer cell adhesion and migration towards nerve cells (snF96.2 or NS20Y) was examined by co-culture assays. Conditioned media (CM) collected from C4-2 cells was cultured with nerve cells (PC-12 or NS20Y) for 48 h followed by qualitative or quantitative neurite outgrowth assay. Our results showed that cancer cells expressing high levels of Snail (LNCaP Snail/C4-2 NS) displayed significantly higher migration adherence to nerve cells, compared to cells with lower levels of Snail (LNCaP Neo/C4-2 Snail shRNA). Additionally, LNCaP Snail or C4-2 NS (Snail-high) CM led to a higher neurite outgrowth compared to the LNCaP Neo or C4-2 Snail shRNA (Snail-low). In conclusion, Snail promotes migration and adhesion to nerve cells, as well as neurite outgrowth via secretion of soluble factors. Therefore, targeting cancer cell interaction with nerves may contribute to halting prostate cancer progression/metastasis.

Immunohistochemical characterization of immune cell infiltration in paediatric and adult Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia commonly affecting children with frequent somatic mutations in MAPK pathway genes including BRAFV600E and MAP2K1. Some studies suggest that LCH cells can recruit and modulate inflammatory cells, which could provide reciprocal survival signals. To characterize the immune profile of infiltrating inflammatory cells, and to clarify their participation in LCH pathogenesis, a detailed immunohistochemical analysis was performed. Fifteen (10 children, 5 adults) LCH cases were assessed through macrophage (CD68 and CD163), mature dendritic cell (mDC; CD83 and CD208), regulatory T cell (Treg; CD4, CD25 and FOXP3) and cytotoxic lymphocyte (CL; CD56, CD57, perforin and granzyme B) immunomarkers. Moreover, lymphocytic and LCH markers were also analysed. All cases were S100, CD1a, CD207 and CD4-positive. Bcl-2 and cyclin D1 expression was observed in 13 of 15 cases. In the immune microenvironment, M2-polarized macrophages and Tregs were the predominant cell populations, followed by significantly (P<.005) smaller levels of mDCs and CLs. Additionally, the number of CD3+cells was significantly higher than that of CD20+cells. In the CD3+cell population, there were a significantly higher number of CD4+cells than CD8+cells. While there were no differences when comparing the paediatric and adult populations, FOXP3+cells were significantly higher in patients with multisystem involvement and treated with chemotherapy, than single-site cases and those without chemotherapy. Our results suggest that M2-polarized macrophages and Treg infiltration can promote LCH development and survival, probably through pro-tumoral, immunosuppressive and/or cytokine-mediated mechanisms. This work highlights the need for further exploration of immune-targeted therapy for LCH.

Reciprocal Signaling between Myeloid Derived Suppressor and Tumor Cells Enhances Cellular Motility and is Mediated by Structural Cues in the Microenvironment.

Myeloid derived suppressor cells (MDSCs) have gained significant attention for their immunosuppressive role in cancer and their ability to contribute to tumor progression and metastasis. Understanding the role of MDSCs in driving cancer cell migration, a process fundamental to metastasis, is essential to fully comprehend and target MDSC-tumor cell interactions. This study employs microfabricated platforms, which simulate the structural cues present in the tumor microenvironment (TME) to elucidate the effects of MDSCs on the migratory phenotype of cancer cells at the single cell level. The results indicate that the presence of MDSCs enhances the motility of cancer-epithelial cells when directional cues (either topographical or spatial) are present. This behavior appears to be independent of cell-cell contact and driven by soluble byproducts from heterotypic interactions between MDSCs and cancer cells. Moreover, MDSC cell-motility is also impacted by the presence of cancer cells and the cancer cell secretome in the presence of directional cues. Epithelial dedifferentiation is the likely mechanism for changes in cancer cell motility in response to MDSCs. These results highlight the biochemical and biostructural conditions under which MDSCs can support cancer cell migration, and could therefore provide new avenues of research and therapy aimed at stemming cancer progression.

Deregulation of miR-27a may contribute to canine fibroblast activation after coculture with a mast cell tumour cell line.

The tumour microenvironment comprises a diverse range of cells, including fibroblasts, immune cells and endothelial cells, along with extracellular matrix. In particular, fibroblasts are of significant interest as these cells are reprogrammed during tumorigenesis to become cancer‐associated fibroblasts (CAFs), which in turn support cancer cell growth. MicroRNAs (miRNAs) have been shown to be involved in this intercellular crosstalk in humans. To assess whether miRNAs are also involved in the activation of fibroblasts in dogs, we cocultured primary canine skin fibroblasts with the canine mast cell tumour cell line C2 directly or with C2‐derived exosomes, and measured differential abundance of selected miRNAs. Expression of the CAF markers alpha‐smooth muscle actin (ACTA2) and stanniocalcin 1 confirmed the activation of our fibroblasts after coculture. We show that fibroblasts displayed significant downregulation of miR‐27a and let‐7 family members. These changes correlated with significant upregulation of predicted target mRNAs. Furthermore, RNA interference knockdown of miR‐27a revealed that cyclin G1 (CCNG1) exhibited negative correlation at the mRNA and protein level, suggesting that CCNG1 is a target of miR‐27a in canine fibroblasts and involved in their activation. Importantly, miR‐27a knockdown itself resulted in fibroblast activation, as demonstrated by the formation of ACTA2 filaments. In addition, interleukin‐6 (IL‐6) was strongly induced in our fibroblasts when cocultured, indicating potential reciprocal signalling. Taken together, our findings are consistent with canine fibroblasts being reprogrammed into CAFs to further support cancer development and that downregulation of miR‐27a may play an important role in the tumour–microenvironment crosstalk.

Reciprocal signaling between mTORC1 and MNK2 controls cell growth and oncogenesis.

eIF4E plays key roles in protein synthesis and tumorigenesis. It is phosphorylated by the kinases MNK1 and MNK2. Binding of MNKs to eIF4G enhances their ability to phosphorylate eIF4E. Here, we show that mTORC1, a key regulator of mRNA translation and oncogenesis, directly phosphorylates MNK2 on Ser74. This suppresses MNK2 activity and impairs binding of MNK2 to eIF4G. These effects provide a novel mechanism by which mTORC1 signaling impairs the function of MNK2 and thereby decreases eIF4E phosphorylation. MNK2[S74A] knock-in cells show enhanced phosphorylation of eIF4E and S6K1 (i.e., increased mTORC1 signaling), enlarged cell size, and increased invasive and transformative capacities. MNK2[Ser74] phosphorylation was inversely correlated with disease progression in human prostate tumors. MNK inhibition exerted anti-proliferative effects in prostate cancer cells in vitro. These findings define a novel feedback loop whereby mTORC1 represses MNK2 activity and oncogenic signaling through eIF4E phosphorylation, allowing reciprocal regulation of these two oncogenic pathways.

MiR-149 Suppresses Breast Cancer Metastasis by Blocking Paracrine Interactions with Macrophages.

Paracrine activation of cells contained in the tumor microenvironment promotes tumor progression and metastasis. In breast cancer, malignant cells recruit and educate macrophages into a M2 tumor-promoting phenotype that supports the metastatic spread of cancer cells. Here, we show that miR-149 functions as a metastasis-suppressing microRNA in breast cancer cells by limiting colony-stimulating factor-1 (CSF1)-dependent recruitment and M2 polarization of macrophages. In lymph node-positive, triple-negative breast cancer (TNBC) tissues, low miR-149 expression correlated with macrophage infiltration and reduced patient survival. By directly targeting CSF1, miR-149 expression in TNBC cell lines (MDA-MB-231 and BT-549) inhibited the recruitment of human monocytic THP-1 cells and primary human macrophages. Furthermore, in macrophages cocultured with MDA-MB-231 cells expressing miR-149, epidermal growth factor (EGF) and amphiregulin expression levels were strongly reduced, resulting in reduced EGF receptor activation in the cancer cells. In vivo, lung metastases developing from orthotopic MDA-MB-231 tumors were reduced by 75% by miR-149 expression, and this was associated with impaired M2 macrophage infiltration of the primary tumors. These data suggest that miR-149 downregulation functionally contributes to breast tumor progression by recruiting macrophages to the tumor and facilitating CSF1 and EGF receptor cross-talk between cancer cells and macrophages. SIGNIFICANCE: These findings contribute to the understanding of tumor-stroma interactions by showing that miR-149 downregulation in TNBC enhances reciprocal growth factor signaling between macrophages and cancer cells, which promotes tumor progression and metastasis. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/6/1330/F1.large.jpg.

Cardiac Neural Crest.

Cardiac neural crest (CNC) cells are pluripotent cells derived from the dorsal neural tube that migrate and contribute to the remodeling of pharyngeal arch arteries and septation of the cardiac outflow tract (OFT). Numerous molecular cascades regulate the induction, specification, delamination, and migration of the CNC. Extensive analyses of the CNC ranging from chick ablation models to molecular biology studies have explored the mechanisms of heart development and disease, particularly involving the OFT and aortic arch (AA) system. Recent studies focus more on reciprocal signaling between the CNC and cells originated from the second heart field (SHF), which are essential for the development of the OFT myocardium, providing new insights into the molecular mechanisms underlying congenital heart diseases (CHDs) and some human syndromes.