miR‐10b expression in breast cancer stem cells supports self‐renewal through negative PTEN regulation and sustained AKT activation

Cancer stem cells (CSCs) are linked to metastasis. Moreover, a discrete group of miRNAs (metastamiRs) has been shown to promote metastasis. Accordingly, we propose that miRNAs that function as metastatic promoters may influence the CSC phenotype. To study this issue, we compared the expression of 353 miRNAs in CSCs enriched from breast cancer cell lines using qRT–PCR analysis. One of the most altered miRNAs was miR‐10b, which is a reported promoter of metastasis and migration. Stable overexpression of miR‐10b in MCF‐7 cells (miR‐10b‐OE cells) promoted higher self‐renewal and expression of stemness and epithelial–mesenchymal transition (EMT) markers. In agreement with these results, inhibiting miR‐10b expression using synthetic antisense RNAs resulted in a decrease in CSCs self‐renewal. Bioinformatics analyses identified several potential miR‐10b mRNA targets, including phosphatase and tensin homolog (PTEN), a key regulator of the PI3K/AKT pathway involved in metastasis, cell survival, and self‐renewal. The targeting of PTEN by miR‐10b was confirmed using a luciferase reporter, qRT–PCR, and Western blot analyses. Lower PTEN levels were observed in CSCs, and miR‐10b depletion not only increased PTEN mRNA and protein expression but also decreased the activity of AKT, a downstream PTEN target kinase. Correspondingly, PTEN knockdown increased stem cell markers, whereas AKT inhibitors compromised the self‐renewal ability of CSCs and breast cancer cell lines overexpressing miR‐10b. In conclusion, miR‐10b regulates the self‐renewal of the breast CSC phenotype by inhibiting PTEN and maintaining AKT pathway activation.     

MicroRNA expression profile of colon cancer stem-like cells in HT29 adenocarcinoma cell line

Increasing evidence has suggested cancer stem cells (CSCs) are considered to be responsible for cancer formation, recurrence, and metastasis. Recently, many studies have also revealed that microRNAs (miRNAs) strongly implicate in regulating self renewal and tumorigenicity of CSCs in human cancers. However, with respect to colon cancer, the role of miRNAs in stemness maintenance and tumorigenicity of CSCs still remains to be unknown. In the present study, we isolated a population of colon CSCs expressing a CD133 surface phenotype from human HT29 colonic adenocarcinoma cell line by Flow Cytometry Cell Sorting. The CD133⁺ cells possess a greater tumor sphere-forming efficiency in vitro and higher tumorigenic potential in vivo. Furthermore, the CD133⁺ cells are endowed with stem/progenitor cells-like property including expression of “stemness” genes involved in Wnt2, BMI1, Oct3/4, Notch1, C-myc and other genes as well as self-renewal and differentiation capacity. Moreover, we investigated the miRNA expression profile of colon CSCs using miRNA array. Consequently, we identified a colon CSCs miRNA signature comprising 11 overexpressed and 8 underexpressed miRNAs, such as miR-429, miR-155, and miR-320d, some of which may be involved in regulation of stem cell differentiation. Our results suggest that miRNAs might play important roles in stemness maintenance of colon CSCs, and analysis of specific miRNA expression signatures may contribute to potential cancer therapy.     

microRNA‐128‐3p overexpression inhibits breast cancer stem cell characteristics through suppression of Wnt signalling pathway by down‐regulating NEK2

Emerging evidence has reported that dysregulation of microRNAs (miRNAs) participated in the development of diverse types of cancers. Our initial microarray‐based analysis identified differentially expressed NEK2 related to breast cancer and predicted the regulatory microRNA‐128‐3p (miR‐128‐3p). Herein, this study aimed to characterize the tumour‐suppressive role of miR‐128‐3p in regulating the biological characteristics of breast cancer stem cells (BCSCs). CD44^＋CD24^?/low cells were selected for subsequent experiments. After verification of the target relationship between miR‐128‐3p and NEK2, the relationship among miR‐128‐3p, NEK2 and BCSCs was further investigated with the involvement of the Wnt signalling pathway. The regulatory effects of miR‐128‐3p on proliferation, migration, invasion and self‐renewal in vitro as well as tumorigenicity in vivo of BCSCs were examined via gain‐ and loss‐of‐function approaches. Highly expressed NEK2 was found in breast cancer based on GSE61304 expression profile. Breast cancer stem cells and breast cancer cells showed a down‐regulation of miR‐128‐3p. Overexpression of miR‐128‐3p was found to inhibit proliferation, migration, invasion, self‐renewal in vitro and tumorigenicity in vivo of BCSCs, which was further validated to be achieved through inhibition of Wnt signalling pathway by down‐regulating NEK2. In summary, this study indicates that miR‐128‐3p inhibits the stem‐like cell features of BCSCs via inhibition of the Wnt signalling pathway by down‐regulating NEK2, which provides a new target for breast cancer treatment.     

Rotavirus‐induced miR‐142‐5p elicits proviral milieu by targeting non‐canonical transforming growth factor beta signalling and apoptosis in cells

MicroRNA (miRNA) expression is significantly influenced by viral infection, because of either host antiviral defences or proviral factors resulting in the modulation of viral propagation. This study was undertaken to identify and analyse the significance of cellular miRNAs during rotavirus (SA11 or KU) infection. Sixteen differentially regulated miRNAs were identified during rotavirus infection of which hsa‐miR‐142‐5p was up‐regulated and validated by quantitative polymerase chain reaction. Exogenous expression of miR‐142‐5p inhibitor resulted in a significant reduction of viral titer indicating proviral role of miR‐142‐5p. Functional studies of hsa‐miR‐142‐5p identified its role in transforming growth factor beta (TGFβ) signalling as TGFβ receptor 2 and SMAD3 were degraded during both hsa‐miR‐142‐5p overexpression and rotavirus infection. TGFβ is induced during rotavirus infection, which may promote apoptosis by activation of non‐canonical pathways in HT29 cells. However, up‐regulated miR‐142‐5p resulted in the inhibition of TGFβ‐induced apoptosis suggesting its anti‐apoptotic function. Rotavirus NSP5 was identified as a regulator of miR‐142‐5p expression. Concurrently, NSP5‐HT29 cells showed inhibition of TGFβ‐induced apoptosis and epithelial to mesenchymal transition by blocking non‐canonical pathways. Overall, the study identified proviral function of hsa‐miR‐142‐5p during rotavirus infection. In addition, modulation of TGFβ‐induced non‐canonical signalling in microsatellite stable colon cancer cells can be exploited for cancer therapeutics.     

Sirtuins at the crossroads of stemness,aging, and cancer

Sirtuins are stress‐responsive proteins that direct various post‐translational modifications (PTMs) and as a result, are considered to be master regulators of several cellular processes. They are known to both extend lifespan and regulate spontaneous tumor development. As both aging and cancer are associated with altered stem cell function, the possibility that the involvement of sirtuins in these events is mediated by their roles in stem cells is worthy of investigation. Research to date suggests that the individual sirtuin family members can differentially regulate embryonic, hematopoietic as well as other adult stem cells in a tissue‐ and cell type‐specific context. Sirtuin‐driven regulation of both cell differentiation and signaling pathways previously involved in stem cell maintenance has been described where downstream effectors involved determine the biological outcome. Similarly, diverse roles have been reported in cancer stem cells (CSCs), depending on the tissue of origin. This review highlights the current knowledge which places sirtuins at the intersection of stem cells, aging, and cancer. By outlining the plethora of stem cell‐related roles for individual sirtuins in various contexts, our purpose was to provide an indication of their significance in relation to cancer and aging, as well as to generate a clearer picture of their therapeutic potential. Finally, we propose future directions which will contribute to the better understanding of sirtuins, thereby further unraveling the full repertoire of sirtuin functions in both normal stem cells and CSCs.     

An integrated analysis to predict micro‐RNAs targeting both stemness and metastasis in breast cancer stem cells

Several evidences support the idea that a small population of tumour cells representing self‐renewal potential are involved in initiation, maintenance, metastasis, and outcomes of cancer therapy. Elucidation of microRNAs/genes regulatory networks activated in cancer stem cells (CSCs) is necessary for the identification of new targets for cancer therapy. The aim of the present study was to predict the miRNAs pattern, which can target both metastasis and self‐renewal pathways using integration of literature and data mining. For this purpose, mammospheres derived from MCF‐7, MDA‐MB231, and MDA‐MB468 were used as breast CSCs model. They had higher migration, invasion, and colony formation potential, with increasing in stemness‐ and EMT‐related genes expression. Our results determined that miR‐204, ‐200c, ‐34a, and ‐10b contemporarily could target both self‐renewal and EMT pathways. This core regulatory of miRNAs could increase the survival rate of breast invasive carcinoma via up‐regulation of OCT4, SOX2, KLF4, c‐MYC, NOTCH1, SNAI1, ZEB1, and CDH2 and down‐regulation of CDH1. The majority of those target genes were involved in the regulation of pluripotency, MAPK, WNT, Hedgehog, p53, and transforming growth factor β pathways. Hence, this study provides novel insights for targeting core regulatory of miRNAs in breast CSCs to target both self‐renewal and metastasis potential and eradication of breast cancer.     

miR‐214 activates TP53 but suppresses the expression of RELA,CTNNB1, and STAT3 in human cervical and colorectal cancer cells

High Mobility Group AT‐hook 1 (HMGA1) was identified as a target of miR‐214 in human cervical and colorectal cancers (CaCx and CRC) in a previous study. While the expression of miR‐214 remains suppressed, HMGA1 behaves as a potent oncogene and plays crucial roles in several aberrant signalling pathways by interacting with intermediates like RELA, CTNNB1, STAT3, and TP53 in CaCx and CRC. Hypothetically, miR‐214 should be able to regulate the stabilization of some of these intermediates through the regulation of HMGA1. This was assessed by ectopically expressing miR‐214 or complementarily, by inhibiting the expression of HMGA1. In promoter luciferase assays, miR‐214 inhibited NF‐κB and Wnt activities but elevated TP53 activity in cancer cells. Further, miR‐214 suppressed the expression of HMGA1, RELA, CTNNB1, and STAT3 while elevating TP53 levels, similar to when small interfering RNA (siRNA) against HMGA1 was used, as revealed by Western blotting. It is suggested that poor expression of miR‐214, commonly reported in CaCx and CRC tissues, may not only result in the sustained expression of HMGA1 but also that of RELA, CTNNB1, and STAT3, and a congruent suppression of TP53 during cancer initiation/progression. These several states are, however, reversed when miR‐214 is reintroduced and could explain the tumour suppressive functions observed in earlier studies. Further studies are, however, required to reveal how microRNA‐mediated regulation of HMGA1 expression may affect individual signalling pathways in CaCx and CRC. Current results reveal that miR‐214 is not only able to regulate the expression of its direct target, HMGA1, but also that of a few signalling intermediates like TP53, RELA, CTNNB1, and STAT3, with which HMGA1 interacts. These intermediates play crucial roles in signalling pathways commonly deregulated in human CaCx and CRC. Hence, it is proposed that miR‐214 might act as a tumour suppressor by regulating several aberrant signalling pathways through HMGA1. This knowledge has the potential to help design novel therapeutic strategies in CaCx and CRC.     

microRNA‐124 inhibits stem‐like properties and enhances radiosensitivity in nasopharyngeal carcinoma cells via direct repression of expression of JAMA

Cancer stem cells (CSCs) are a source of tumour recurrence in patients with nasopharyngeal carcinoma (NPC); however, the function of microRNA‐124 (miR‐124) in NPC CSCs has not been clearly defined. In this study, we investigated the role of miR‐124 in NPC CSCs. qRT‐PCR was performed to measure miR‐124 expression in NPC tissues and cell lines and the effects of miR‐124 on stem‐like properties and radiosensitivity of NPC cells measured. Luciferase reporter assays and rescue experiments were used to investigate the interaction of miR‐124 with the 3′UTR of junctional adhesion molecule A (JAMA). Finally, we examined the effects of miR‐124 in an animal model and clinical samples. Down‐regulation of miR‐124 was detected in cancer tissues and was inversely associated with tumour stage and lymph node metastasis. Overexpression of miR‐124 inhibited stemness properties and enhanced radiosensitivity of NPC cells in vitro and in vivo via targeting JAMA. Up‐regulation of miR‐124 was correlated with superior overall survival of patients with NPC. Our study demonstrates that miR‐124 can inhibit stem‐like properties and enhance radiosensitivity by directly targeting JAMA in NPC. These findings provide novel insights into the molecular mechanisms underlying therapy failure in NPC.     

The functions and targets of miR‐212 as a potential biomarker of cancer diagnosis and therapy

Cancer is a major health problem worldwide. An increasing number of researchers are studying the diagnosis, therapy and mechanisms underlying the development and progression of cancer. The study of noncoding RNA has attracted a lot of attention in recent years. It was found that frequent alterations of miRNA expression not only have various functions in cancer but also that miRNAs can act as clinical markers of diagnosis, stage and progression of cancer. MiR‐212 is an important example of miRNAs involved in cancer. According to recent studies, miR‐212 may serve as an oncogene or tumour suppressor by influencing different targets or pathways during the oncogenesis and the development and metastasis of cancer. Its deregulation may serve as a marker for the diagnosis or prognosis of cancer. In addition, it was recently reported that miR‐212 was related to the sensitivity or resistance of cancer cells to chemotherapy or radiotherapy. Here, we summarize the current understanding of miR‐212 functions in cancer by describing the relevant signalling pathways and targets. The role of miR‐212 as a biomarker and its therapeutic potential in cancer is also described. The aim of this review was to identify new methods for the diagnosis and treatment of human cancers.     

Developmental signaling pathways in cancer stem cells of solid tumors

Background

The intricate regulation of several signaling pathways is essential for embryonic development and adult tissue homeostasis. Cancers commonly display aberrant activity within these pathways. A population of cells identified in several cancers, termed cancer stem cells (CSCs) show similar properties to normal stem cells and evidence suggests that altered developmental signaling pathways play an important role in maintaining CSCs and thereby the tumor itself.

Scope of review

This review will focus on the roles of the Notch, Wnt and Hedgehog pathways in the brain, breast and colon cancers. We describe the roles these pathways play in normal tissue homeostasis through the regulation of stem cell fate in these three tissues, and the experimental evidence indicating that the role of these pathways in cancers of these is directly linked to CSCs.

Major conclusions

A large body of evidence is accumulating to indicate that the deregulation of Notch, Wnt and Hedgehog pathways play important roles in both normal and cancer stem cells. We are only beginning to understand how these pathways interact, how they are coordinated during normal development and adult tissue homeostasis, and how they are deregulated during cancer. However, it is becoming increasingly clear that if we are to target CSCs therapeutically, it will likely be necessary to develop combination therapies.

General significance

If CSCs are the driving force behind tumor maintenance and growth then understanding the molecular mechanisms regulating CSCs is essential. Such knowledge will contribute to better targeted therapies that could significantly enhance cancer treatments and patient survival. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

MicroRNAs as Regulators of Neural Stem Cell-Related Pathways in Glioblastoma Multiforme

MicroRNAs are endogenous non-coding small RNAs that have been described as highly conserved regulators of gene expression. They are involved in cancer and in the regulation of neural development and stem cell function. Recent studies suggest that a small subpopulation of cancer stem cells (CSCs) has the capacity to repopulate solid tumours such as glioblastoma (GBM), drive malignant progression and mediate radio- and chemoresistance. GBM-derived CSCs share the fundamental stem cell properties of self-renewal and multipotency with neural stem cells (NSCs) and may be regulated by miRNAs. In this review, we will summarize the current knowledge regarding the role of miRNAs in GBM development with a focus on the regulation of GBM-CSCs. We propose a list of miRNAs that could serve as molecular classifiers for GBMs and/or as promising therapeutic targets for such brain tumours.     

EGF signalling pathway regulates colon cancer stem cell proliferation and apoptosis

Objectives

Cancer stem cells (CSCs) compose a subpopulation of cells within a tumour that can self‐renew and proliferate. Growth factors such as epidermal growth factor (EGF) and basic fibroblast growth factor (b‐FGF) promote cancer stem cell proliferation in many solid tumours. This study assesses whether EGF, bFGF and IGF signalling pathways are essential for colon CSC proliferation and self‐renewal.

Material and methods

Colon CSCs were cultured in serum‐free medium (SFM) with one of the following growth factors: EGF, bFGF or IGF. Characteristics of CSC gene expression were evaluated by real time PCR. Tumourigenicity of CSCs was determined using a xenograft model in vivo. Effects of EGF receptor inhibitors, Gefitinib and PD153035, on CSC proliferation, apoptosis and signalling were evaluated using fluorescence‐activated cell sorting and western blotting.

Results

Colon cancer cell HCT116 transformed to CSCs in SFM. Compared to other growth factors, EGF was essential to support proliferation of CSCs that expressed higher levels of progenitor genes (Musashi‐1, LGR5) and lower levels of differential genes (CK20). CSCs promoted more rapid tumour growth than regular cancer cells in xenografts. EGFR inhibitors suppressed proliferation and induced apoptosis of CSCs by inhibiting autophosphorylation of EGFR and downstream signalling proteins, such as Akt kinase, extracellular signal‐regulated kinase 1/2 (ERK 1/2).

Conclusions

This study indicates that EGF signalling was essential for formation and maintenance of colon CSCs. Inhibition of the EGF signalling pathway may provide a useful strategy for treatment of colon cancer.     

The functional role of microRNAs in alcoholic liver injury

The function of microRNAs (miRNAs) during alcoholic liver disease (ALD) has recently become of great interest in biological research. Studies have shown that ALD associated miRNAs play a crucial role in the regulation of liver‐inflammatory agents such as tumour necrosis factor‐alpha (TNF‐α), one of the key inflammatory agents responsible for liver fibrosis (liver scarring) and the critical contributor of alcoholic liver disease. Lipopolysaccharide (LPS), a component of the cell wall of gram‐negative bacteria, is responsible for TNF‐α release by Kupffer cells. miRNAs are the critical mediators of LPS signalling in Kupffer cells, hepatocytes and hepatic stellate cells. Certain miRNAs, in particular miR‐155 and miR‐21, show a positive correlation in up‐regulation of LPS signalling when they are exposed to ethanol. ALD is related to enhanced gut permeability that allows the levels of LPS to increase, leads to increased secretion of TNF‐α by the Kupffer cells and subsequently promotes alcoholic liver injury through specific miRNAs. Meanwhile, two of the most frequently dysregulated miRNAs in steatohepatitis, miR‐122 and miR‐34a are the critical mediators in ethanol/LPS activated survival signalling during ALD. In this review, we summarize recent findings regarding the experimental and clinical aspects of functions of specific microRNAs, focusing mainly on inflammation and cell survival after ethanol/LPS treatment, and advances on the role of circulating miRNAs in human alcoholic disorders.     

Myc‐regulated microRNAs attenuate embryonic stem cell differentiation

Myc proteins are known to have an important function in stem cell maintenance. As Myc has been shown earlier to regulate microRNAs (miRNAs) involved in proliferation, we sought to determine whether c‐Myc also affects embryonic stem (ES) cell maintenance and differentiation through miRNAs. Using a quantitative primer‐extension PCR assay we identified miRNAs, including, miR‐141, miR‐200, and miR‐429 whose expression is regulated by c‐Myc in ES cells, but not in the differentiated and tumourigenic derivatives of ES cells. Chromatin immunoprecipitation analyses indicate that in ES cells c‐Myc binds proximal to genomic regions encoding the induced miRNAs. We used expression profiling and seed homology to identify genes specifically downregulated both by these miRNAs and by c‐Myc. We further show that the introduction of c‐Myc‐induced miRNAs into murine ES cells significantly attenuates the downregulation of pluripotency markers on induction of differentiation after withdrawal of the ES cell maintenance factor LIF. In contrast, knockdown of the endogenous miRNAs accelerate differentiation. Our data show that in ES cells c‐Myc acts, in part, through a subset of miRNAs to attenuate differentiation.