Embryonic MicroRNA-369 Controls Metabolic Splicing Factors and Urges Cellular Reprograming.

Noncoding microRNAs inhibit translation and lower the transcript stability of coding mRNA, however miR-369 s, in aberrant silencing genomic regions, stabilizes target proteins under cellular stress. We found that in vitro differentiation of embryonic stem cells led to chromatin methylation of histone H3K4 at the miR-369 region on chromosome 12qF in mice, which is expressed in embryonic cells and is critical for pluripotency. Proteomic analyses revealed that miR-369 stabilized translation of pyruvate kinase (Pkm2) splicing factors such as HNRNPA2B1. Overexpression of miR-369 stimulated Pkm2 splicing and enhanced induction of cellular reprogramming by induced pluripotent stem cell factors, whereas miR-369 knockdown resulted in suppression. Furthermore, immunoprecipitation analysis showed that the Argonaute complex contained the fragile X mental retardation-related protein 1 and HNRNPA2B1 in a miR-369-depedent manner. Our findings demonstrate a unique role of the embryonic miR-369-HNRNPA2B1 axis in controlling metabolic enzyme function, and suggest a novel pathway linking epigenetic, transcriptional, and metabolic control in cell reprogramming.

A mammosphere formation RNAi screen reveals that ATG4A promotes a breast cancer stem-like phenotype.

INTRODUCTION: Breast cancer stem cells are suspected to be responsible for tumour recurrence, metastasis formation as well as chemoresistance. Consequently, great efforts have been made to understand the molecular mechanisms underlying cancer stem cell maintenance. In order to study these rare cells in-vitro, they are typically enriched via mammosphere culture. Here we developed a mammosphere-based negative selection shRNAi screening system suitable to analyse the involvement of thousands of genes in the survival of cells with cancer stem cell properties. METHODS: We describe a sub-population expressing the stem-like marker CD44(+)/CD24(-/low) in SUM149 that were enriched in mammospheres. To identify genes functionally involved in the maintenance of the sub-population with cancer stem cell properties, we targeted over 5000 genes by RNAi and tested their ability to grow as mammospheres. The identified candidate ATG4A was validated in mammosphere and soft agar colony formation assays. Further, we evaluated the influence of ATG4A expression on the sub-population expressing the stem-like marker CD44(+)/CD24(low). Next, the tumorigenic potential of SUM149 after up- or down-regulation of ATG4A was examined by xenograft experiments. RESULTS: Using this method, Jak-STAT as well as cytokine signalling were identified to be involved in mammosphere formation. Furthermore, the autophagy regulator ATG4A was found to be essential for the maintenance of a sub-population with cancer stem cell properties and to regulate breast cancer cell tumourigenicity in vivo. CONCLUSION: In summary, we present a high-throughput screening system to identify genes involved in cancer stem cell maintenance and demonstrate its utility by means of ATG4A.

Effect of in vivo administration of reprogramming factors in the mouse liver.

Cancer is initiated by the transformation of stem cells or progenitor cells via a dedifferentiation process that leads to cancer stem cells; however, the process involves the activation of growth-promoting oncogenes and the inactivation of growth-constraining tumor suppressor genes. The introduction of defined factors, such as those encoded by c-Myc, Sox2, Oct3/4 and Klf4, in normal somatic cells results in their dedifferentiation into induced pluripotent stem (iPS) cells. We previously reported that these defined factors induced the development of induced multipotent cancer (iPC) cells from gastrointestinal cancer cells by reducing tumor aggressiveness. Previous studies indicated that although reprogramming may be facilitated by p53 inhibition, gain-of-function oncogenic mutations in p53 and oncogenic mutations in Kras-stimulated tumorigenic activity, and their roles in vivo are imperfectly understood. Hence, in the present study, the effect of direct injection of a Sendai virus (SeV) vector encoding four defined factors in vivo was studied using various backgrounds of transgenic and knockout mice, and was compared with that of direct injection of microRNAs (miRNAs) diluted with cationic lipid. The in vivo imaging data revealed transformation hot spots for p53 deficiency or conditional activation of mutant Kras, and the sizes were concordant with those in immuno-deficient NOD/SCID and uPA-NOG mice, as well as larger compared with those in the control mice. Overall, the present data on in vivo reprogramming indicated that Kras activation may facilitate the effect of cellular reprogramming in normal liver cells, and the effect of Kras activation is more apparent than that of tumor suppressor p53 deficiency. The results also revealed that immunodeficiency may increase the effect of reprogramming, presumably by blocking the immunosurveillance of transformed cells. These findings provide a rationale for further studies to develop a therapeutic approach involving direct in vivo reprogramming.

microRNA-based cancer cell reprogramming technology.

Epigenetic modifications play crucial roles in cancer initiation and development. Complete reprogramming can be achieved through the introduction of defined biological factors such as Oct4, Sox2, Klf4, and cMyc into mouse and human fibroblasts. Introduction of these transcription factors resulted in the modification of malignant phenotype behavior. Recent studies have shown that human and mouse somatic cells can be reprogrammed to become induced pluripotent stem cells using forced expression of microRNAs, which completely eliminates the need for ectopic protein expression. Considering the usefulness of RNA molecules, microRNA-based reprogramming technology may have future applications in regenerative and cancer medicine.

Transcriptomic study of dormant gastrointestinal cancer stem cells.

We previously discovered the coexistence of dormant and proliferating cancer stem cells (CSCs) in gastrointestinal cancer, which leads to chemoradiation resistance. CD13-/CD90+ proliferating liver CSCs are sensitive to chemotherapy, and CD13+/CD90- dormant CSCs have a limited proliferation ability, survive in hypoxic areas with reduced oxidative stress, and relapse and metastasize to other organs. In such CD13+ dormant cells, non-homologous end-joining, an error-prone repair mechanism, is dominant after DNA damage, whereas high-fidelity homologous recombination is apparent in CD13- proliferating cells, suggesting the significance of dormancy as an essential protective mechanism of therapy resistance. However, this mechanism may also play a role in the generation and accumulation of heterogeneity during cancer progression, although the exact mechanism remains to be understood. Through transcriptomic study, we elucidated the underlying epigenetic mechanism for malignant behavior of dormant CSCs, i.e., simultaneous activation of several pathways including EZH2- and TP53-related proteins in response to microRNA101, suggesting that a pharmacogenomic approach would open an era to novel molecular targeting cancer therapy.

Dicer 1, ribonuclease type III modulates a reprogramming effect in colorectal cancer cells.

Complete cell reprogramming can be achieved by the introduction of specific transcription factors, Oct4 [also known as POU class 5 homeobox 1 (Pou5f1)]; sex-determining region Y (SRY)-box 2 (Sox2); Kruppel-like factor 4 (Klf4); and myelocytomatosis viral oncogene homolog (c-Myc), into terminally differentiated mouse somatic fibroblasts. This reprogramming process may be accelerated or suppressed by various factors, including microRNAs (miRNAs). Introduction of these transcription factors or miRNAs considerably modifies the malignant phenotype of cancer cells. We studied the effect of introducing these transcription factors into two distinct colorectal cancer (CRC) cell lines, HCT116 and DLD-1, in the presence and absence of Dicer 1, ribonuclease type III (Dicer1), a critical miRNA processing enzyme. We assessed cell reprogramming based on the number of cells exhibiting alkaline phosphatase staining and an increase in embryonic stem cell-like gene expression, indicating the return of cells to an immature state. Dicer1-deficient CRC cells showed a reduced number of alkaline phosphatase-positive reprogrammed cells than wild-type (WT) cells. Before reprogramming, endogenous expression of an immature carbohydrate epitope, TRA-1-60, was high in Dicer1-deficient CRC cells, whereas after reprogramming, the expression of this epitope was increased in Dicer1-sufficient more than in Dicer1-deficient CRC cells. Our data demonstrate the critical role of miRNAs in the reprogramming process and determination of a differentiated phenotype of CRC cells.

Cancer stem cell theory in gastrointestinal malignancies: recent progress and upcoming challenges.

A growing body of evidence supports the notion that malignant tumors are heterogeneous and contain diverse subpopulations of cells with unique characteristics including the ability to initiate a tumor and metastasize. This phenomenon might be explained by the so-called cancer stem cell (CSC) theory. Recent technological developments have allowed a deeper understanding and characterization of CSCs. Even though the application of this theory to hematopoietic malignancies and solid tumors holds promise for new ways to treat cancer, it also brings some skepticism. Efficacious therapeutic approaches targeting the CSC population should be explored to overcome therapeutic failure and improve patient outcomes. This review will focus on the intrinsic and extrinsic regulation of CSCs, as well as the development of therapeutic approaches against CSCs, predominantly focusing on gastrointestinal malignancies.

Reprogramming of mouse and human cells to pluripotency using mature microRNAs.

Induced pluripotent stem cells (iPSCs) can be generated from differentiated human and mouse somatic cells using transcription factors such as Oct4, Sox2, Klf4, and c-Myc. It is possible to augment the reprogramming process with chemical compounds, but issues related to low reprogramming efficiencies and, with a number of protocols, residual vector sequences, remain to be resolved. We show here that it is possible to reprogram mouse and human cells to pluripotency by direct transfection of mature double-stranded microRNAs (miRNAs). Our approaches use a combination of mir-200c plus mir-302 s and mir-369 s family miRNAs. Because this reprogramming method does not require vector-based gene transfer, it holds significant potential for biomedical research and regenerative medicine.