miR-22 represses cancer progression by inducing cellular senescence

Cellular senescence acts as a barrier to cancer progression, and microRNAs (miRNAs) are thought to be potential senescence regulators. However, whether senescence-associated miRNAs (SA-miRNAs) contribute to tumor suppression remains unknown. Here, we report that miR-22, a novel SA-miRNA, has an impact on tumorigenesis. miR-22 is up-regulated in human senescent fibroblasts and epithelial cells but down-regulated in various cancer cell lines. miR-22 overexpression induces growth suppression and acquisition of a senescent phenotype in human normal and cancer cells. miR-22 knockdown in presenescent fibroblasts decreased cell size, and cells became more compact. miR-22-induced senescence also decreases cell motility and inhibits cell invasion in vitro. Synthetic miR-22 delivery suppresses tumor growth and metastasis in vivo by inducing cellular senescence in a mouse model of breast carcinoma. We confirmed that CDK6, SIRT1, and Sp1, genes involved in the senescence program, are direct targets of miR-22. Our study provides the first evidence that miR-22 restores the cellular senescence program in cancer cells and acts as a tumor suppressor.     

Activin and TGFβ regulate expression of the microRNA-181 family to promote cell migration and invasion in breast cancer cells

MicroRNA-181 (miR-181) is a multifaceted miRNA that has been implicated in many cellular processes such as cell fate determination and cellular invasion. While miR-181 is often overexpressed in human tumors, a direct role for this miRNA in breast cancer progression has not yet been characterized. In this study, we found this miRNA to be regulated by both activin and TGFβ. While we found no effect of miR-181 modulation on activin/TGFβ-mediated tumor suppression, our data clearly indicate that miR-181 plays a critical and prominent role downstream of two growth factors, in mediating their pro-migratory and pro-invasive effects in breast cancer cells miR-181 acts as a metastamir in breast cancer. Thus, our findings define a novel role for miR-181 downstream of activin/TGFβ in regulating their tumor promoting functions. Having defined miR-181 as a critical regulator of tumor progression in vitro, our results thus, highlight miR-181 as an important potential therapeutic target in breast cancer.     

Plasma MicroRNA Panel for Minimally Invasive Detection of Breast Cancer

Over the last few years, circulating microRNAs (miRNAs) have emerged as promising novel and minimally invasive markers for various diseases, including cancer. We already showed that certain miRNAs are deregulated in the plasma of breast cancer patients when compared to healthy women. Herein we have further explored their potential to serve as breast cancer early detection markers in blood plasma. Circulating miR-127-3p, miR-376a and miR-652, selected as candidates from a miRNA array-based screening, were found to be associated with breast cancer for the first time (n = 417). Further we validated our previously reported circulating miRNAs (miR-148b, miR-376c, miR-409-3p and miR-801) in an independent cohort (n = 210) as elevated in the plasma of breast cancer patients compared to healthy women. We described, for the first time in breast cancer, an over-representation of deregulated miRNAs (miR-127-3p, miR-376a, miR-376c and miR-409-3p) originating from the chromosome 14q32 region. The inclusion of patients with benign breast tumors enabled the observation that miR-148b, miR-652 and miR-801 levels are even elevated in the plasma of women with benign tumors when compared to healthy controls. Furthermore, an analysis of samples stratified by cancer stage demonstrated that miR-127-3p, miR-148b, miR-409-3p, miR-652 and miR-801 can detect also stage I or stage II breast cancer thus making them attractive candidates for early detection. Finally, ROC curve analysis showed that a panel of these seven circulating miRNAs has substantial diagnostic potential with an AUC of 0.81 for the detection of benign and malignant breast tumors, which further increased to 0.86 in younger women (up to 50 years of age).     

Programmed cell death 4 (PDCD4) is an important functional target of the microRNA miR-21 in breast cancer cells

MicroRNAs are emerging as important regulators of cancer-related processes. The miR-21 microRNA is overexpressed in a wide variety of cancers and has been causally linked to cellular proliferation, apoptosis, and migration. Inhibition of mir-21 in MCF-7 breast cancer cells causes reduced cell growth. Using array expression analysis of MCF-7 cells depleted of miR-21, we have identified mRNA targets of mir-21 and have shown a link between miR-21 and the p53 tumor suppressor protein. We furthermore found that the tumor suppressor protein Programmed Cell Death 4 (PDCD4) is regulated by miR-21 and demonstrated that PDCD4 is a functionally important target for miR-21 in breast cancer cells.     

miR-223促进小鼠胚胎成纤维细胞复制性衰老

微小RNA（MicroRNAs（或miRNAs）是作为强大的基因表达调控子,广泛参与多种生命过程,在细胞衰老进程中的作用也日益受到关注。miR-223是一个典型的抑癌基因,可显著抑制细胞增殖能力。此外,miR-223与阿尔茨海默症、心血管疾病以及类风湿性关节炎等衰老相关疾病的发生发展密切相关。尽管如此,miR-223在细胞衰老进程中的作用及其分子机制尚未见报道。本研究通过连续传代建立了小鼠胚胎成纤维细胞（MEF细胞）的复制性衰老模型,并利用荧光定量qRT-PCR检测发现,miR-223在衰老MEF细胞中的表达水平显著上调。随后,通过转染miR-223模拟物Agomir-223在MEF细胞中过表达miR-223,结果显示过表达miR-223可显著促进MEF细胞的衰老表型并抑制其增殖能力,而抑制miR-223的表达可延缓MEF细胞的复制性衰老进程。进一步利用生物信息学方法预测获得多个miR-223的候选衰老相关靶基因,包括Rasa1、Ddit4和Smad1等。然而双萤光素酶报告系统结果显示,miR-223并不显著影响其萤光强度,表明它们很可能并不是miR-223的下游靶基因。综上所述,miR-223可显著促进MEF细胞复制性衰老,然而其调节细胞衰老进程的分子机制依然有待深入研究。     

BMP-6 inhibits cell proliferation by targeting microRNA-192 in breast cancer

Although bone morphogenetic protein-6 (BMP-6) has been identified as a tumor suppressor associated with breast cancer differentiation and metastasis, the potential roles of BMP-6 in regulating cell cycle progression have not been fully examined. In the present study, we provide the novel finding that induction of BMP-6 in MDA-MB-231 breast cancer cells significantly inhibits cell proliferation by decreasing the number of cells in S phase of the cell cycle, resulting in inhibition of tumorigenesis in a nude mouse xenograft model. Further investigation indicated that BMP-6 up-regulates the expression of microRNA-192 (miR-192) in MDA-MB-231 cells. Elevated expression of miR-192 caused cell growth arrest, which is similar to the effect of BMP-6 induction. Importantly, depletion of endogenous miR-192 by miRNA inhibition significantly attenuated BMP-6-mediated repression of cell cycle progression. In breast cancer tissue, miR-192 expression is significantly down-regulated in tumor samples and positively correlates with the expression of BMP-6, demonstrating the inhibitory effect of BMP-6 on cell proliferation through miR-192 regulation. Additionally, using the RT² Profiler PCR Array, retinoblastoma 1 (RB1) was identified as a direct target of the BMP-6/miR-192 pathway in regulating cell proliferation in breast cancer. In conclusion, we have identified an important role for BMP-6/miR-192 signaling in the regulation of cell cycle progression in breast cancer. Furthermore, BMP-6/miR-192 was expressed at low levels in breast cancer specimens, indicating that this pathway might represent a promising therapeutic target for breast cancer treatment.     

MicroRNA 133B targets pro-survival molecules MCL-1 and BCL2L2 in lung cancer

Lung cancer is the most frequent cause of cancer-related death in this country for men and women. MicroRNAs (miRNAs) are a family of small non-coding RNAs (approximately 21-25 nt long) capable of targeting genes for either degradation of mRNA or inhibition of translation. We identified aberrant expression of 41 miRNAs in lung tumor versus uninvolved tissue. MiR-133B had the lowest expression of miRNA in lung tumor tissue (28-fold reduction) compared to adjacent uninvolved tissue. We identified two members of the BCL-2 family of pro-survival molecules (MCL-1 and BCL2L2 (BCLw)) as predicted targets of miR-133B. Selective over-expression of miR-133B in adenocarcinoma (H2009) cell lines resulted in reduced expression of both MCL-1 and BCL2L2. We then confirmed that miR-133B directly targets the 3′UTRs of both MCL-1 and BCL2L2. Lastly, over-expression of miR-133B induced apoptosis following gemcitabine exposure in these tumor cells. To our knowledge, this represents the first observation of decreased expression of miR-133B in lung cancer and that it functionally targets members of the BCL-2 family.     

The miR-15a-miR-16-1 cluster controls prostate cancer by targeting multiple oncogenic activities

MicroRNAs (miRNAs) are noncoding small RNAs that repress protein translation by targeting specific messenger RNAs. miR-15a and miR-16-1 act as putative tumor suppressors by targeting the oncogene BCL2. These miRNAs form a cluster at the chromosomal region 13q14, which is frequently deleted in cancer. Here, we report that the miR-15a and miR-16-1 cluster targets CCND1 (encoding cyclin D1) and WNT3A, which promotes several tumorigenic features such as survival, proliferation and invasion. In cancer cells of advanced prostate tumors, the miR-15a and miR-16 level is significantly decreased, whereas the expression of BCL2, CCND1 and WNT3A is inversely upregulated. Delivery of antagomirs specific for miR-15a and miR-16 to normal mouse prostate results in marked hyperplasia, and knockdown of miR-15a and miR-16 promotes survival, proliferation and invasiveness of untransformed prostate cells, which become tumorigenic in immunodeficient NOD-SCID mice. Conversely, reconstitution of miR-15a and miR-16-1 expression results in growth arrest, apoptosis and marked regression of prostate tumor xenografts. Altogether, we propose that miR-15a and miR-16 act as tumor suppressor genes in prostate cancer through the control of cell survival, proliferation and invasion. These findings have therapeutic implications and may be exploited for future treatment of prostate cancer.     

miR-7 and miR-218 epigenetically control tumor suppressor genes RASSF1A and Claudin-6 by targeting HoxB3 in breast cancer

Many microRNAs have been implicated as key regulators of cellular growth and differentiation and have been found to dysregulate proliferation in human tumors, including breast cancer. Cancer-linked microRNAs also alter the epigenetic landscape by way of DNA methylation and post-translational modifications of histones. Aberrations in Hox gene expression are important for oncogene or tumor suppressor during abnormal development and malignancy. Although recent studies suggest that HoxB3 is critical in breast cancer, the putative role(s) of microRNAs impinging on HoxB3 is not yet fully understood. In this study, we found that the expression levels of miR-7 and miR-218 were strongly and reversely associated with HoxB3 expression. Stable overexpression of miR-7 and miR-218 was accompanied by reactivation of tumor suppressor genes including RASSF1A and Claudin-6 by means of epigenetic switches in DNA methylation and histone modification, giving rise to inhibition of the cell cycle and clone formation of breast cancer cells. The current study provides a novel link between overexpression of collinear Hox genes and multiple microRNAs in human breast malignancy.     

Down-Regulation of eIF4GII by miR-520c-3p Represses Diffuse Large B Cell Lymphoma Development

Deregulation of the translational machinery is emerging as a critical contributor to cancer development. The contribution of microRNAs in translational gene control has been established however; the role of microRNAs in disrupting the cap-dependent translation regulation complex has not been previously described. Here, we established that elevated miR-520c-3p represses global translation, cell proliferation and initiates premature senescence in HeLa and DLBCL cells. Moreover, we demonstrate that miR-520c-3p directly targets translation initiation factor, eIF4GII mRNA and negatively regulates eIF4GII protein synthesis. miR-520c-3p overexpression diminishes cells colony formation and reduces tumor growth in a human xenograft mouse model. Consequently, downregulation of eIF4GII by siRNA decreases translation, cell proliferation and ability to form colonies, as well as induces cellular senescence. In vitro and in vivo findings were further validated in patient samples; DLBCL primary cells demonstrated low miR-520c-3p levels with reciprocally up-regulated eIF4GII protein expression. Our results provide evidence that the tumor suppressor effect of miR-520c-3p is mediated through repression of translation while inducing senescence and that eIF4GII is a key effector of this anti-tumor activity.     

Targeting Stat3 induces senescence in tumor cells and elicits prophylactic and therapeutic immune responses against breast cancer growth mediated by NK cells and CD4+ T cells

Aberrant Stat3 activation and signaling contribute to malignant transformation by promoting cell cycle progression, inhibiting apoptosis, and mediating tumor immune evasion. Stat3 inhibition in tumor cells induces the expression of chemokines and proinflammatory cytokines, so we proposed to apply Stat3-inhibited breast cancer cells as a source of immunogens to induce an antitumor immune response. Studies were performed in two murine breast cancer models in which Stat3 is activated: progestin-dependent C4HD cells and 4T1 cells. We immunized BALB/c mice with irradiated cancer cells previously transfected with a dominant-negative Stat3 vector (Stat3Y705F) in either a prophylactic or a therapeutic manner. Prophylactic administration of breast cancer cells transfected with Stat3Y705F (Stat3Y705F-breast cancer cells) inhibited primary tumor growth compared with administration of empty vector-transfected cells in both models. In the 4T1 model, 50% of the challenged mice were tumor free, and the incidence of metastasis decreased by 90%. In vivo assays of C4HD tumors showed that the antitumor immune response involves the participation of CD4(+) T cells and cytotoxic NK cells. Therapeutic immunization with Stat3Y705F-breast cancer cells inhibited tumor growth, promoted tumor cell differentiation, and decreased metastasis. Furthermore, inhibition of Stat3 activation in breast cancer cells induced cellular senescence, contributing to their immunogenic phenotype. In this work, we provide preclinical proof of concept that ablating Stat3 signaling in breast cancer cells results in an effective immunotherapy against breast cancer growth and metastasis. Moreover, our findings showing that Stat3 inactivation results in induction of a cellular senescence program disclose a potential mechanism for immunotherapy research.