The microRNA-302-367 cluster suppresses the proliferation of cervical carcinoma cells through the novel target AKT1

The miR-302-367 cluster is specifically expressed in human embryonic stem cells and has been shown to convert human somatic cells into induced pluripotent stem cells. Here, we investigated the role of the miR-302-367 cluster in cervical carcinoma. The cluster was not endogenously expressed in cervical cancer cells, and its ectopic expression did not reprogram the cervical cancer cells to an embryonic stem cell-like state. However, ectopic expression of the miR-302-367 cluster in HeLa and SiHa cervical cancer cells inhibited cell proliferation and tumor formation by blocking the G1/S cell cycle transition. We identified a new cell cycle regulatory pathway in which the miR-302-367 cluster directly down-regulated both cyclin D1 and AKT1 and indirectly up-regulated p27^Kip1 and p21^Cip1, leading to the suppression of cervical cancer cell proliferation. Our findings suggest that the miR-302-367 cluster may be used as a therapeutic reagent for the treatment of cervical carcinoma.     

MicroRNA-302/367 cluster功能的研究进展

MircroRNA (miRNA)是一段长度约为22个nt的小型非编码RNA,广泛存在于真核生物中,具有调节基因表达的作用。对miRNA的鉴定、功能分析和调控机理研究已成为当今生物领域的热点。miR-302/367cluster属于胚胎干细胞特异性细胞周期调控miRNAs家族成员(embryonic stem cell-specific cell cycle-regulating family of microRNAs,ESCC miRNAs),通常由5个成员miR-302a、miR-302b、miR-302c、miR-302d及miR-367组成,大多分布在脊椎动物中。研究表明,该miRNAs簇对细胞多种生理过程起重要调控作用,如人胚胎干细胞(hESCs)多能性的维持、自我更新等。本研究概述了miRNA的合成及作用机理,ESCC miRNAs促进体细胞再程序化,并总结了miR-302/367 cluster在细胞周期调控、表观遗传修饰及一些细胞信号转导途径中的作用,为采用该类miRNAs诱导体细胞再程序化为iPS细胞(Induced pluripotent stem cells)提供一定的理论基础。     

Vitamin C counteracts miR-302/367-induced reprogramming of human breast cancer cells and restores their invasive and proliferative capacity

Epigenetic reprogramming by embryonic stem cell-specific miR-302/367 cluster has shown some tumor suppressive effects in cancer cells of different tissues such as skin, colon, and cervix. Vitamin C has been known as a reprogramming enhancer of human and mouse somatic cells. In this study, first we aimed to investigate whether exogenous induction of miR-302/367 in breast cancer cells shows the same tumor suppressive effects previously observed in other cancer cells lines, and whether vitamin C can enhance reprogramming of breast cancer cells and also improve the tumor suppressive function of miR-302/367 cluster. Overexpression of miR-302/367 cluster in MDA-MB-231 and SK-BR-3 breast cancer cells upregulated expression of miR-302/367 members and also some core pluripotency factors including OCT4A, SOX2 and NANOG, induced mesenchymal to epithelial transition, suppressed invasion, proliferation, and induced apoptosis in the both cell lines. However, treatment of the miR-302/367 transfected cells with vitamin C suppressed the expression of pluripotency factors and augmented the tumorigenicity of the breast cancer cells by restoring their proliferative and invasive capacity and compromising the apoptotic effect of miR-302/367. Supplementing the culture medium with vitamin C downregulated expression of TET1 gene which seems to be the reason behind the negative impact of vitamin C on the reprogramming efficiency of miR-302/367 cluster and its anti-tumor effects. Therefore application of vitamin C may not always serve as a reprogramming enhancer depending on its switching function on TET1. This phenomenon should be carefully considered when considering a reprogramming strategy for tumor suppression.     

CXCL12 mediates trophic interactions between endothelial and tumor cells in glioblastoma

Emerging evidence suggests endothelial cells (EC) play a critical role in promoting Glioblastoma multiforme (GBM) cell proliferation and resistance to therapy. The molecular basis for GBM-EC interactions is incompletely understood. We hypothesized that the chemokine CXCL12 and its receptor CXCR4 could mediate direct interactions between GBM cells and tumor-associated endothelial cells and that disruption of this interaction might be the molecular basis for the anti-tumor effects of CXCR4 antagonists. We investigated this possibility in vivo and in an in vitro co-culture model that incorporated extracellular matrix, primary human brain microvascular ECs (HBMECs) and either an established GBM cell line or primary GBM specimens. Depletion of CXCR4 in U87 GBM cells blocked their growth as intracranial xenografts indicating that tumor cell CXCR4 is required for tumor growth in vivo. In vitro, co-culture of either U87 cells or primary GBM cells with HBMECs resulted in their co-localization and enhanced GBM cell growth. Genetic manipulation of CXCL12 expression and pharmacological inhibition of its receptors CXCR4 and CXCR7 revealed that the localizing and trophic effects of endothelial cells on GBM cells were dependent upon CXCL12 and CXCR4. These findings indicate that the CXCL12/CXCR4 pathway directly mediates endothelial cell trophic function in GBMs and that inhibition of CXCL12-CXCR4 signaling may uniquely target this activity. Therapeutic disruption of endothelial cell trophic functions could complement the structural disruption of anti-angiogenic regimens and, in combination, might also improve the efficacy of radiation and chemotherapy in treating GBMs.     

HEDGEHOG-GLI1 signaling regulates human glioma growth, cancer stem cell self-renewal, and tumorigenicity

Cancer stem cells are rare tumor cells characterized by their ability to self-renew and to induce tumorigenesis. They are present in gliomas and may be responsible for the lethality of these incurable brain tumors. In the most aggressive and invasive type, glioblastoma multiforme (GBM), an average of about one year spans the period between detection and death [1]. The resistence of gliomas to current therapies may be related to the existence of cancer stem cells [2-6]. We find that human gliomas display a stemness signature and demonstrate that HEDGEHOG (HH)-GLI signaling regulates the expression of stemness genes in and the self-renewal of CD133(+) glioma cancer stem cells. HH-GLI signaling is also required for sustained glioma growth and survival. It displays additive and synergistic effects with temozolomide (TMZ), the current chemotherapeutic agent of choice. TMZ, however, does not block glioma stem cell self-renewal. Finally, interference of HH-GLI signaling with cyclopamine or through lentiviral-mediated silencing demonstrates that the tumorigenicity of human gliomas in mice requires an active pathway. Our results reveal the essential role of HH-GLI signaling in controlling the behavior of human glioma cancer stem cells and offer new therapeutic possibilities.     

Human Cytomegalovirus Gene Expression in Long-Term Infected Glioma Stem Cells

The most common adult primary brain tumor, glioblastoma (GBM), is characterized by fifteen months median patient survival and has no clear etiology. We and others have identified the presence of human cytomegalovirus (HCMV) gene products endogenously expressed in GBM tissue and primary cells, with a subset of viral genes being consistently expressed in most samples. Among these viral genes, several have important oncomodulatory properties, regulating tumor stemness, proliferation, immune evasion, invasion and angiogenesis. These findings lead us to hypothesize that a specific HCMV gene signature may be associated with GBM pathogenesis. To investigate this hypothesis, we used glioma cell lines and primary glioma stem-like cells (GSC) infected with clinical and laboratory HCMV strains and measured relative viral gene expression levels along several time points up to 15 weeks post-infection. While HCMV gene expression was detected in several infected glioma lines through week 5 post-infection, only HCMV-infected GSC expressed viral gene products 15 weeks post-infection. Efficiency of infection across time was higher in GSC compared to cell lines. Importantly, HCMV-infected GSC outlived their uninfected counterparts, and this extended survival was paralleled by increased tumorsphere frequency and upregulation of stemness regulators, such as SOX2, p-STAT3, and BMX (a novel HCMV target identified in this study). Interleukin 6 (IL-6) treatment significantly upregulated HCMV gene expression in long-term infected glioma cultures, suggesting that pro-inflammatory signaling in the tumor milieu may further augment HCMV gene expression and subsequent tumor progression driven by viral-induced cellular signaling. Together, our data support a critical role for long-term, low-level HCMV infection in promoting survival, stemness, and proliferation of GSC that could significantly contribute to GBM pathogenesis.     

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.     

What Do We Learn from Spheroid Culture Systems? Insights from Tumorspheres Derived from Primary Colon Cancer Tissue

Due to their self-renewal and tumorigenic properties, tumor-initiating cells (TICs) have been hypothesized to be important targets for colorectal cancer (CRC). However the study of TICs is hampered by the fact that the identification and culturing of TICs is still a subject of extensive debate. Floating three-dimensional spheroid cultures (SC) that grow in serum-free medium supplemented with growth factors are supposed to be enriched in TICs. We generated SC from fresh clinical tumor specimens and compared them to SC isolated from CRC cell-lines as well as to adherent differentiated counterparts. Patient-derived SC display self-renewal capacity and can induce serial transplantable tumors in immuno-deficient mice, which phenotypically resemble the tumor of origin. In addition, the original tumor tissue and established SC retain several similar CRC-relevant mutations. Primary SC express key stemness proteins such as SOX2, OCT4, NANOG and LGR5 and importantly show increased chemoresistance ability compared to their adherent differentiated counterparts and to cell line-derived SC. Strikingly, cells derived from spheroid or adherent differentiating culture conditions displayed similar self-renewal capacity and equally formed tumors in immune-deficient mice, suggesting that self-renewal and tumor-initiation capacity of TICs is not restricted to phenotypically immature spheroid cells, which we describe to be highly plastic and able to reacquire stem-cell traits even after long differentiation processes. Finally, we identified two genes among a sphere gene expression signature that predict disease relapse in CRC patients. Here we propose that SC derived from fresh patient tumor tissue present interesting phenotypic features that may have clinical relevance for chemoresistance and disease relapse and therefore represent a valuable tool to test for new CRC-therapies that overcome drug resistance.     

Resveratrol suppresses tumorigenicity and enhances radiosensitivity in primary glioblastoma tumor initiating cells by inhibiting the STAT3 axis

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. Patients diagnosed with GBM have a poor prognosis, and it has been reported that tumor malignancy and GBM recurrence are promoted by STAT3 signaling. As resveratrol (RV), a polyphenol in grapes, is reported to be a potent and non-toxic cancer-preventive compound, the aim of this study was to investigate the therapeutic effect and molecular mechanisms of RV on GBM-derived radioresistant tumor initiating cells (TIC). Firstly, our results showed that primary GBM-CD133(+) TIC presented high tumorigenic and radiochemoresistant properties as well as increased protein levels of phosphorylated STAT3. We consistently observed that treatment with shRNA-STAT3 (sh-STAT3) or AG490, a STAT3 inhibitor, significantly inhibited the cancer stem-like cell properties and radioresistance of GBM-CD133(+) in vitro and in vivo. Furthermore, treatment of GBM-CD133(+) with 100 μM RV induced apoptosis and enhanced radiosensitivity by suppressing STAT3 signaling. Microarray results suggested that RV or AG490 inhibited the stemness gene signatures of GBM-CD133(+) and facilitated the differentiation of GBM-CD133(+) into GBM-CD133(-) or astrocytoma cells. Finally, xenotransplant experiments indicated that RV or sh-STAT3 therapy could significantly improve the survival rate and synergistically enhance the radiosensitivity of radiation-treated GBM-TIC. In summary, RV can reduce in vivo tumorigenicity and enhance the sensitivity of GBM-TIC to radiotherapies through the STAT3 pathway.     

miR-139-5p reverses stemness maintenance and metastasis of colon cancer stem-like cells by targeting E2-2

Colon cancer stem cells (CCSCs) stand for a critical subpopulation of colon cancer cells that possess self-renewal and multilineage differentiation potentials and drive tumorigenicity. Due to their impact on treatment tolerance, CCSCs have been a hot research topic in the past few years. We have previously reported that miR-139-5p is a vital tumor repressive noncoding RNA whose level decreases in the clinical colon cancer samples with the increase of tumor malignancy. This research discovered that miR-139-5p targets the Wnt/β-catenin/TCF7L2 downstream effector E2-2 in CCSCs. E2-2 is a pivot molecule in the negative feedback loop of miR-139-5p/Wnt/β-catenin/TCF7L2. Its small interfering RNA reverses the stemness maintenance and epithelial-mesenchymal transition of colon cancer CSCs. This study provides a theoretical foundation for the clinical diagnosis and medical treatment of recurrent or metastatic colon cancer with miR-139-5p and its target E2-2.