Downregulation of miRNA-200c links breast cancer stem cells with normal stem cells.

Human breast tumors contain a breast cancer stem cell (BCSC) population with properties reminiscent of normal stem cells. We found 37 microRNAs that were differentially expressed between human BCSCs and nontumorigenic cancer cells. Three clusters, miR-200c-141, miR-200b-200a-429, and miR-183-96-182 were downregulated in human BCSCs, normal human and murine mammary stem/progenitor cells, and embryonal carcinoma cells. Expression of BMI1, a known regulator of stem cell self-renewal, was modulated by miR-200c. miR-200c inhibited the clonal expansion of breast cancer cells and suppressed the growth of embryonal carcinoma cells in vitro. Most importantly, miR-200c strongly suppressed the ability of normal mammary stem cells to form mammary ducts and tumor formation driven by human BCSCs in vivo. The coordinated downregulation of three microRNA clusters and the similar functional regulation of clonal expansion by miR-200c provide a molecular link that connects BCSCs with normal stem cells.

Adipocyte regulation of cancer stem cells.

Cancer stem cells (CSCs) are a highly tumorigenic subpopulation of the cancer cells within a tumor that drive tumor initiation, progression, and therapy resistance. In general, stem cell niche provides a specific microenvironment in which stem cells are present in an undifferentiated and self-renewable state. CSC niche is a specialized tumor microenvironment for CSCs which provides cues for their maintenance and propagation. However, molecular mechanisms for the CSC-niche interaction remain to be elucidated. We have revealed that adipsin (complement factor D) and its downstream effector hepatocyte growth factor are secreted from adipocytes and enhance the CSC properties in breast cancers in which tumor initiation and progression are constantly associated with the surrounding adipose tissue. Considering that obesity, characterized by excess adipose tissue, is associated with an increased risk of multiple cancers, it is reasonably speculated that adipocyte-CSC interaction is similarly involved in many types of cancers, such as pancreas, colorectal, and ovarian cancers. In this review, various molecular mechanisms by which adipocytes regulate CSCs, including secretion of adipokines, extracellular matrix production, biosynthesis of estrogen, metabolism, and exosome, are discussed. Uncovering the roles of adipocytes in the CSC niche will propose novel strategies to treat cancers, especially those whose progression is linked to obesity.

Local states of chromatin compaction at transcription start sites control transcription levels.

The 'open' and 'compact' regions of chromatin are considered to be regions of active and silent transcription, respectively. However, individual genes produce transcripts at different levels, suggesting that transcription output does not depend on the simple open-compact conversion of chromatin, but on structural variations in chromatin itself, which so far have remained elusive. In this study, weakly crosslinked chromatin was subjected to sedimentation velocity centrifugation, which fractionated the chromatin according to its degree of compaction. Open chromatin remained in upper fractions, while compact chromatin sedimented to lower fractions depending on the level of nucleosome assembly. Although nucleosomes were evenly detected in all fractions, histone H1 was more highly enriched in the lower fractions. H1 was found to self-associate and crosslinked to histone H3, suggesting that H1 bound to H3 interacts with another H1 in an adjacent nucleosome to form compact chromatin. Genome-wide analyses revealed that nearly the entire genome consists of compact chromatin without differences in compaction between repeat and non-repeat sequences; however, active transcription start sites (TSSs) were rarely found in compact chromatin. Considering the inverse correlation between chromatin compaction and RNA polymerase binding at TSSs, it appears that local states of chromatin compaction determine transcription levels.

Monocyte-Derived miRNA-1914-5p Attenuates IL-1ß-Induced Monocyte Adhesion and Transmigration.

Atherosclerosis can lead to cardiovascular and cerebrovascular diseases. Atherosclerotic plaque formation is promoted by the accumulation of inflammatory cells. Therefore, modulating monocyte recruitment represents a potential therapeutic strategy. In an inflammatory state, the expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) is upregulated in endothelial cells. We previously reported that miR-1914-5p in endothelial cells suppresses interleukin (IL)-1ß-induced ICAM-1 expression and monocyte adhesion to endothelial cells. However, whether monocyte miR-1914-5p affects monocyte recruitment is unclear. In this study, IL-1ß decreased miR-1914-5p expression in a human monocyte cell line. Moreover, miR-1914-5p inhibition enhanced adhesion to endothelial cells with the upregulation of macrophage-1 antigen (Mac-1), a counter-ligand to ICAM-1. Transmigration through the endothelial layer was also promoted with the upregulation of monocyte chemotactic protein-1 (MCP-1). Furthermore, a miR-1914-5p mimic suppressed IL-1ß-induced monocyte adhesion and transmigration in monocytes with Mac-1 and MCP-1 downregulation. Further investigation of miR-1914-5p in monocytes could lead to the development of novel diagnostic markers and therapeutic strategies for atherosclerosis.

Maternal fructose intake predisposes rat offspring to metabolic disorders via abnormal hepatic programming.

Given that fructose consumption has increased by more than 10-fold in recent decades, it is possible that excess maternal fructose consumption causes harmful effects in the next generation. This study attempted to elucidate the mechanism of the harmful effects of excessive maternal fructose intake from the perspective of offspring liver function. Female rats during gestation and lactation were fed water containing fructose, and their offspring were fed normal water. We attempted to elucidate the mechanism of fructose-induced transgenerational toxicity by conducting a longitudinal study focusing on hepatic programming prior to disease onset. Impaired Insulin resistance and decreased high-density lipoprotein-cholesterol levels were observed at 160 days of age. However, metabolic disorders were not observed in 60-day-old offspring. Microarray analysis of 60-day-old offspring livers showed the reduction of hepatic insulin-like growth factor-1 (Igf1) mRNA expression. This reduction continued until the rats were aged 160 days and attenuated Igf1 signaling. Hepatic microRNA-29 (miR-29a) and miR-130a, which target Igf1 mRNA, were also found to be upregulated. Interestingly, these miRNAs were upregulated in the absence of metabolic disorder. In this study, we found that maternal fructose intake resulted in dysregulated expression of Igf1 and its target miRNAs in the offspring liver, and that these offspring were more likely to develop metabolic disorders. Abnormal hepatic programming induced by an imbalanced maternal nutritional environment is maintained throughout life, implying that it may contribute to metabolic disorders.

MicroRNA-93 targets WASF3 and functions as a metastasis suppressor in breast cancer.

Cancer cells with cancer stem cell (CSC) properties initiate both primary tumor formation and metastases at distant sites. Acquisition of CSC properties is highly associated with epigenetic alterations, including those mediated by microRNAs (miRNAs). We have previously established the breast cancer patient-derived tumor xenograft (PDX) mouse model in which CSC marker CD44+ cancer cells formed spontaneous microscopic metastases in the liver. In this PDX mouse, we found that the expression levels of 3 miRNAs (miR-25, miR-93, and miR-106b) in the miR-106b-25 cluster were much lower in the CD44+ human cancer cells metastasized to the liver than those at the primary site. Constitutive overexpression of miR-93 suppressed invasive ability and 3D-organoid formation capacity of breast cancer cells in vitro and significantly suppressed their metastatic ability to the liver in vivo. Wiskott-Aldrich syndrome protein family member 3 (WASF3), a regulator of both cytoskeleton remodeling and CSC properties, was identified as a functional target of miR-93: overexpression of miR-93 reduced the protein level of WASF3 in breast cancer cells and WASF3 rescued the miR-93-mediated suppression of breast cancer cell invasion. These findings suggest that miR-93 functions as a metastasis suppressor by suppressing both invasion ability and CSC properties in breast cancers.

Upregulation of S100A10 in metastasized breast cancer stem cells.

Metastatic progression remains the major cause of death in human breast cancer. Cancer cells with cancer stem cell (CSC) properties drive initiation and growth of metastases at distant sites. We have previously established the breast cancer patient-derived tumor xenograft (PDX) mouse model in which CSC marker CD44+ cancer cells formed spontaneous microscopic metastases in the liver. In this PDX mouse, the expression levels of S100A10 and its family proteins were much higher in the CD44+ cancer cells metastasized to the liver than those at the primary site. Knockdown of S100A10 in breast cancer cells suppressed and overexpression of S100A10 in breast cancer PDX cells enhanced their invasion abilities and 3D organoid formation capacities in vitro. Mechanistically, S100A10 regulated the matrix metalloproteinase activity and the expression levels of stem cell-related genes. Finally, constitutive knockdown of S100A10 significantly reduced their metastatic ability to the liver in vivo. These findings suggest that S100A10 functions as a metastasis promoter of breast CSCs by conferring both invasion ability and CSC properties in breast cancers.

Maternal fructose-induced oxidative stress occurs viaTfam and Ucp5 epigenetic regulation in offspring hippocampi.

Fructose consumption is rising globally, but maternal high fructose intake might adversely affect offspring. Our previous report demonstrated that excess maternal fructose intake impairs hippocampal function in offspring, indicating that the hippocampi of offspring are highly sensitive to maternal fructose. Here, we examined the effect of maternal high fructose on mitochondrial physiology and uncoupling protein (UCP) expression. Rat dams received a 20% fructose solution during gestation and lactation. Immediately after weaning, offspring hippocampi were isolated. Maternal high fructose consumption attenuated the mitochondrial O2 consumption rate and stimulated lipid hydroperoxide production in the hippocampi of offspring. Reduced Ucp5 and mitochondrial transcription factor A (Tfam) mRNA levels were also observed after maternal exposure to fructose. We assessed the promoter regions of both genes and found that this treatment enhanced DNA methylation levels. In addition, luciferase assays showed that this DNA methylation could reduce the transcription of both genes. Chromatin immunoprecipitation analysis demonstrated that specificity protein 1 binding to the Ucp5 promoter regions was reduced by DNA methylation. In addition, Ucp5 knockdown induced the up-regulation of reactive oxygen species levels in a rat brain glioma cell line, whereas reduced O2 consumption was observed with Tfam knockdown. Maternal high fructose intake thus induces reduced O2 oxygen consumption and increases oxidative stress in offspring, at least partly through epigenetic mechanisms involving Ucp5 and Tfam.-Yamada, H., Munetsuna, E., Yamazaki, M., Mizuno, G., Sadamoto, N., Ando, Y., Fujii, R., Shiogama, K., Ishikawa, H., Suzuki, K., Shimono, Y., Ohashi, K., Hashimoto, S. Maternal fructose-induced oxidative stress occurs viaTfam and Ucp5 epigenetic regulation in offspring hippocampi.

Hippo vs. Crab: tissue-specific functions of the mammalian Hippo pathway.

The Hippo signaling pathway is a vital suppressor of tumorigenesis that is often inactivated in human cancers. In normal cells, the Hippo pathway is triggered by external forces such as cell crowding, or changes to the extracellular matrix or cell polarity. Once activated, Hippo signaling down-regulates transcription supported by the paralogous cofactors YAP1 and TAZ. The Hippo pathway's functions in normal and cancer biology have been dissected by studies of mutant mice with null or conditional tissue-specific mutations of Hippo signaling elements. In this review, we attempt to systematically summarize results that have been gleaned from detailed in vivo characterizations of these mutants. Our goal is to describe the physiological roles of Hippo signaling in several normal organ systems, as well as to emphasize how disruption of the Hippo pathway, and particularly hyperactivation of YAP1/TAZ, can be oncogenic.

Expression of programmed death-1 in sentinel lymph nodes of breast cancer.

BACKGROUND AND OBJECTIVES: To explore whether lymphocytes in sentinel lymph nodes (SLNs) are highly exposed to tumor neoantigens and thus express high level of programmed death 1 (PD-1), we examined PD-1 expression in SLNs and non-sentinel regional lymph nodes (non-SLNs) in breast cancer. METHODS: We performed PD-1 immunohistochemistry in two cohorts: 40 metastasis-negative SLNs including 10 patients for each subtype (luminal A-like, luminal B-like, HER2, and triple negative breast cancer [TNBC]); and 25 pairs of metastasis-positive SLNs and non-SLNs (10 luminal A-like, 10 luminal B-like, and 5 TNBC). RESULTS: Among 40 metastasis-negative SLNs, 34 and 6 samples were PD-1 intensity grade 1 (low) and 2 (high), respectively. PD-1 intensity correlated with PD-1-positive lymphocyte numbers (P = 0.005); TNBC had the highest PD-1 lymphocyte numbers among all subtypes. The median PD-1-positive lymphocyte number was higher in SLNs than non-SLNs. In most cases, more lymphocytes in SLNs expressed PD-1 than those in non-SLNs (P < 0.0001). CONCLUSIONS: TNBC had the greatest PD-1 expression among all subtypes, and metastasis-positive SLNs had more PD-1-positive lymphocytes than downstream non-SLNs. These data suggested that lymphocytes in SLNs are activated following exposure to tumor neoantigens and thus tumor specific, and could be utilized as a biomarker platform.

A Novel Nectin-mediated Cell Adhesion Apparatus That Is Implicated in Prolactin Receptor Signaling for Mammary Gland Development.

Mammary gland development is induced by the actions of various hormones to form a structure consisting of collecting ducts and milk-secreting alveoli, which comprise two types of epithelial cells known as luminal and basal cells. These cells adhere to each other by cell adhesion apparatuses whose roles in hormone-dependent mammary gland development remain largely unknown. Here we identified a novel cell adhesion apparatus at the boundary between the luminal and basal cells in addition to desmosomes. This apparatus was formed by the trans-interaction between the cell adhesion molecules nectin-4 and nectin-1, which were expressed in the luminal and basal cells, respectively. Nectin-4 of this apparatus further cis-interacted with the prolactin receptor in the luminal cells to enhance the prolactin-induced prolactin receptor signaling for alveolar development with lactogenic differentiation. Thus, a novel nectin-mediated cell adhesion apparatus regulates the prolactin receptor signaling for mammary gland development.

Glucose metabolism-targeted therapy and withaferin A are effective for epidermal growth factor receptor tyrosine kinase inhibitor-induced drug-tolerant persisters.

In pathway-targeted cancer drug therapies, the relatively rapid emergence of drug-tolerant persisters (DTPs) substantially limits the overall therapeutic benefit. However, little is known about the roles of DTPs in drug resistance. In this study, we investigated the features of epidermal growth factor receptor-tyrosine kinase inhibitor-induced DTPs and explored a new treatment strategy to overcome the emergence of these DTPs. We used two EGFR-mutated lung adenocarcinoma cell lines, PC9 and II-18. They were treated with 2 µM gefitinib for 6, 12, or 24 days or 6 months. We analyzed the mRNA expression of the stem cell-related markers by quantitative RT-PCR and the expression of the cellular senescence-associated proteins. Then we sorted DTPs according to the expression pattern of CD133 and analyzed the features of sorted cells. Finally, we tried to ablate DTPs by glucose metabolism targeting therapies and a stem-like cell targeting drug, withaferin A. Drug-tolerant persisters were composed of at least two types of cells, one with the properties of cancer stem-like cells (CSCs) and the other with the properties of therapy-induced senescent (TIS) cells. The CD133high cell population had CSC properties and the CD133low cell population had TIS properties. The CD133low cell population containing TIS cells showed a senescence-associated secretory phenotype that supported the emergence of the CD133high cell population containing CSCs. Glucose metabolism inhibitors effectively eliminated the CD133low cell population. Withaferin A effectively eliminated the CD133high cell population. The combination of phloretin and withaferin A effectively suppressed gefitinib-resistant tumor growth.

Regulation of CD44 expression and focal adhesion by Golgi phosphatidylinositol 4-phosphate in breast cancer.

CD44, a transmembrane receptor, is expressed in the standard or variant form and plays a critical role in tumor progression and metastasis. This protein regulates cell adhesion and migration in breast cancer cells. We previously reported that phosphatidylinositol-4-phosphate (PI(4)P) at the Golgi regulates cell migration and invasion in breast cancer cell lines. In this study, we showed that an increase in PI(4)P levels at the Golgi by knockdown of PI(4)P phosphatase SAC1 increased the expression of standard CD44, variant CD44, and ezrin/radixin phosphorylation and enhanced the formation of focal adhesions mediated by CD44 and ezrin/radixin in MCF7 and SK-BR-3 cells. In contrast, knockdown of PI 4-kinase IIIß in highly invasive MDA-MB-231 cells decreased these factors. These results suggest that SAC1 expression and PI(4)P at the Golgi are important in tumor progression and metastasis and are potential prognostic markers of breast cancers.

Suppression of the TGF-ß1-induced protein expression of SNAI1 and N-cadherin by miR-199a.

MicroRNA miR-199a is clustered with miR-214 on chromosome 1 and its expression is up-regulated by various factors that are associated with epithelial-to-mesenchymal transition (EMT), such as a transcriptional repressor Twist1 and transforming growth factor (TGF)-ß. miR-199a is either up-regulated or down-regulated in a variety of cancers, although EMT is associated with the progression of cancer. We found here that miR-199a suppressed the translation of SNAI1, a transcriptional repressor that plays a role in EMT, by targeting the sequence within the 3'UTR of the SNAI1 mRNA, and reduced the protein level of SNAI1. miR-199a increased the protein level of claudin-1 in both the TGF-ß1-treated and -untreated cells at least partly by decreasing the protein level of SNAI1, a transcriptional repressor for claudin-1. In addition, miR-199a targeted the sequence within the 3'UTR of the N-cadherin mRNA and suppressed the TGF-ß1-induced increase in the protein level of N-cadherin in a manner independent of SNAI1. These results indicate that miR-199a suppresses the TGF-ß1-induced protein expression of SNAI1 and N-cadherin.

Absence of primary cilia in cell cycle-arrested human breast cancer cells.

Previous studies using cultured cells showed that primary cilia are present in quiescent cells, but are absent in proliferating cells. We studied here the relationship between the presence or absence of primary cilia and the cell cycle arrest of normal epithelial cells and cancer cells in the human normal breast and breast cancer tissues. In normal breast tissues, although most epithelial cells were nonproliferating as estimated by the immunofluorescence staining of the proliferation marker Ki-67, primary cilia were present only in 20-40% of the epithelial cells. In breast cancer tissues, primary cilia were not observed in any of the breast cancer cells. Furthermore, primary cilia were hardly observed in the nonproliferating cancer cells in the orthotopic and metastatic human breast cancer xenograft tumors in mice. These results indicate that the absence of primary cilia does not necessarily represent the proliferating phases of normal epithelial cells and cancer cells.

[Effect of Natural Killer Cell Infiltration on the Growth of Breast Cancer Patient-Derived Tumor Xenografts].

Natural killer (NK) cells, a component of the innate immunity, play important roles in tumor suppression. In this study, three human breast cancer patient-derived tumor xenografts (PDXs), established by the transplantation of surgical specimens, were passaged in immunodeficient NOD/SCID mice or NSG mice, that further lacks NK cell activity. The intensity of the relative growth suppression between NOD/SCID and NSG mice was clearly different depending on the PDX lines, and it was associated with the intensities of the CD49b-positive NK cell infiltration in the PDX tumor tissues. However, no obvious association was observed between the mRNA expression levels of the NK cell ligands in the PDX tumor cells and the intensity of NK cell infiltration into the PDX tumors. These results suggest that the suppressive effect of NK cells on the growth of breast cancer PDX is highly variable depending on the PDX lines. Further studies are needed to elucidate the molecular mechanism of NK cell infiltration in PDX tumors.

[Breast cancer stem cell research utilizing clinical specimens].

The existence of cancer stem cells in solid tumors was first reported in 2003 based on the analyses of human breast cancers. Analyses of clinical specimens are especially important for the advancement of human cancer stem cell research. For example, the analyses of breast cancer stem cells directly isolated from human breast cancer specimens identified the microRNAs that are involved in the regulation of human breast cancer stem cells. In addition, human breast cancer xenograft is an attractive model to analyze cancer stem cells, as well as cancer metastases that have profound effect on the long-term survival of breast cancer patients. We will present the current status of human breast cancer stem cell research that utilizes clinical specimens.

Reduction of the ST6 ß-galactosamide α-2,6-sialyltransferase 1 (ST6GAL1)-catalyzed sialylation of nectin-like molecule 2/cell adhesion molecule 1 and enhancement of ErbB2/ErbB3 signaling by microRNA-199a.

Nectin-like molecule 2 (Necl-2)/cell adhesion molecule 1 (CADM1) is shown to be down-regulated by the promoter hypermethylation and/or loss of heterozygosity at chromosome 11q23.2 in many types of cancers, including lung and breast cancers, and is proposed to serve as a tumor suppressor. However, the incidence of these epigenetic and genetic abnormalities of Necl-2 is 30-60% in these cancers, and other mechanisms for the suppression of Necl-2 are presumed to be present. We previously showed that Necl-2 interacts in cis with ErbB3 and suppresses the heregulin (HRG)-induced ErbB2/ErbB3 signaling for cell movement and death. We studied here the relationship between Necl-2 and microRNA-199a (miR-199a) that is up-regulated or down-regulated in a variety of cancers. miR-199a did not directly target the Necl-2 mRNA or affect its mRNA level in human lung cancer A549 cells and human embryonic kidney HEK293 cells. Necl-2 was at least sialylated by the sialyltransferase ST6 ß-galactosamide α-2,6-sialyltransferase 1 (ST6GAL1). miR-199a targeted ST6GAL1 and reduced both the sialylation and the protein level of Necl-2. In addition, miR-199a enhanced the HRG-induced ErbB2/ErbB3 signaling. These results indicate that the suppressive role of Necl-2 in the HRG-induced ErbB2/ErbB3 signaling is regulated by miR-199a at least through the reduction of the ST6GAL1-catalyzed sialylation of Necl-2 and/or through the reduction of the protein level of Necl-2 presumably by the protein degradation.

Interaction of Necl-4/CADM4 with ErbB3 and integrin α6 ß4 and inhibition of ErbB2/ErbB3 signaling and hemidesmosome disassembly.

Nectin-like molecule 4 (Necl-4)/CADM4, a transmembrane cell-cell adhesion molecule with three Ig-like domains, was shown to serve as a tumor suppressor, but its mode of action has not been elucidated. In this study, we showed that Necl-4 interacted in cis with ErbB3 through their extracellular regions, recruited PTPN13 and inhibited the heregulin-induced activation of the ErbB2/ErbB3 signaling. In addition, we extended our previous finding that Necl-4 interacts in cis with integrin α6 ß4 through their extracellular regions and found that Necl-4 inhibited the phorbol ester-induced disassembly of hemidesmosomes. These results indicate that Necl-4 serves as a tumor suppressor by inhibiting the ErbB2/ErbB3 signaling and hemidesmosome disassembly.

miR-214 and hypoxia down-regulate Necl-2/CADM1 and enhance ErbB2/ErbB3 signaling.

Necl-2/CADM1 is down-regulated by the promoter hypermethylation and/or the loss of heterozygosity at chromosome 11q23.2 in many types of cancers and serves as a tumor suppressor by interacting in cis with ErbB3 and suppressing the ligand-induced ErbB2/ErbB3 signaling for cell movement and death. However, the incidence of these epigenetic and genetic abnormalities of Necl-2 is 30-60% in these cancers. We investigated here other mechanisms that down-regulate Necl-2. miR-214, that is frequently up-regulated in a variety of cancers, targeted the 3'UTR of the Necl-2 mRNA directly, suppressed the translation of Necl-2 and enhanced the ligand-induced ErbB2/ErbB3 signaling in human colon cancer Caco-2 cells. Hypoxia reduced the Necl-2 protein level in a manner independent of miR-214 or hypoxia-inducible factor-1α in Caco-2 cells. These results indicate that miR-214 and hypoxia are novel regulators that down-regulate Necl-2 and enhance ErbB2/ErbB3 signaling.