Synergism between the mTOR inhibitor rapamycin and FAK down-regulation in the treatment of acute lymphoblastic leukemia.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is an aggressive malignant disorder of lymphoid progenitor cells in both children and adults. Although improvements in contemporary therapy and development of new treatment strategies have led to dramatic increases in the cure rate in children with ALL, the relapse rate remains high and the prognosis of relapsed childhood ALL is poor. Molecularly targeted therapies have emerged as the leading treatments in cancer therapy. Multi-cytotoxic drug regimens have achieved success, yet many studies addressing targeted therapies have focused on only one single agent. In this study, we attempted to investigate whether the effect of the mammalian target of rapamycin (mTOR) inhibitor rapamycin is synergistic with the effect of focal adhesion kinase (FAK) down-regulation in the treatment of ALL. METHODS: The effect of rapamycin combined with FAK down-regulation on cell proliferation, the cell cycle, and apoptosis was investigated in the human precursor B acute lymphoblastic leukemia cells REH and on survival time and leukemia progression in a non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mouse model. RESULTS: When combined with FAK down-regulation, rapamycin-induced suppression of cell proliferation, G0/G1 cell cycle arrest, and apoptosis were significantly enhanced. In addition, REH cell-injected NOD/SCID mice treated with rapamycin and a short-hairpin RNA (shRNA) to down-regulate FAK had significantly longer survival times and slower leukemia progression compared with mice injected with REH-empty vector cells and treated with rapamycin. Moreover, the B-cell CLL/lymphoma-2 (BCL-2) gene family was shown to be involved in the enhancement, by combined treatment, of REH cell apoptosis. CONCLUSIONS: FAK down-regulation enhanced the in vitro and in vivo inhibitory effects of rapamycin on REH cell growth, indicating that the simultaneous targeting of mTOR- and FAK-related pathways might offer a novel and powerful strategy for treating ALL.

MIR125B1 represses the degradation of the PML-RARA oncoprotein by an autophagy-lysosomal pathway in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is characterized by the t(15;17)-associated PML-RARA fusion gene. We have previously found that MIR125B1 is highly expressed in patients with APL and may be associated with disease pathogenesis; however, the mechanism by which MIR125B1 exerts its oncogenic potential has not been fully elucidated. Here, we demonstrated that MIR125B1 abundance correlates with the PML-RARA status. MIR125B1 overexpression enhanced PML-RARA expression and inhibited the ATRA-induced degradation of the PML-RARA oncoprotein. RNA-seq analysis revealed a direct link between the PML-RARA degradation pathway and MIR125B1-arrested differentiation. We further demonstrated that the MIR125B1-mediated blockade of PML-RARA proteolysis was regulated via an autophagy-lysosomal pathway, contributing to the inhibition of APL differentiation. Furthermore, we identified DRAM2 (DNA-damage regulated autophagy modulator 2), a critical regulator of autophagy, as a novel target that was at least partly responsible for the function of MIR125B1 involved in autophagy. Importantly, the knockdown phenotypes for DRAM2 are similar to the effects of overexpressing MIR125B1 as impairment of PML-RARA degradation, inhibition of autophagy, and myeloid cell differentiation arrest. These effects of MIR125B1 and its target DRAM2 were further confirmed in an APL mouse model. Thus, MIR125B1 dysregulation may interfere with the effectiveness of ATRA-mediated differentiation through an autophagy-dependent pathway, representing a novel potential APL therapeutic target.

A distinct set of long non-coding RNAs in childhood MLL-rearranged acute lymphoblastic leukemia: biology and epigenetic target.

Long non-coding RNAs (lncRNAs) have been recently found to be pervasively transcribed in human genome and link to diverse human diseases. However, the expression patterns and regulatory roles of lncRNAs in hematopoietic malignancies have not been reported. Here, we carried out a genome-wide lncRNA expression study in MLL-rearranged acute lymphoblastic leukemia (MLL-r ALL) and established lncRNA/messenger RNA coexpression networks to gain insight into the biological roles of these dysregulated lncRNAs. We detected a number of lncRNAs that were differentially expressed in MLL-r ALL samples compared with MLL-r wild-type and identified unique lncRNA expression patterns between MLL-r subtypes with different translocations as well as between infant MLL-r ALL with other MLL-r ALL patients, suggesting that they might be served as novel biomarkers for the disease. Importantly, several lncRNAs that correspond with membrane protein genes, including a lysosome-associated membrane protein, were identified. No such link between the membrane proteins and MLL-r leukemia has been reported previously. Impressively, the functional analysis showed that several lncRNAs corresponded to the expression of MLL-fusion protein target genes, including HOXA9, MEIS1, etc., while some other associated with histone-related functions or membrane proteins. Further experiments characterize the effect of some lncRNAs on MLL-r leukemia apoptosis and proliferation as the function of the coexpressed HOXA gene cluster. Finally, a set of lncRNAs epigenetically regulated by H3K79 methylation were also discovered. These findings may provide novel insights into the mechanisms of lncRNAs involved in the initiation of MLL-r leukemia. This is the first study linking lncRNAs to leukemogenesis.

miR-133b, a muscle-specific microRNA, is a novel prognostic marker that participates in the progression of human colorectal cancer via regulation of CXCR4 expression.

BACKGROUND: MicroRNA-133b (miR-133b), which is a muscle-specific microRNA, has been reported to be downregulated in human colorectal carcinoma (CRC) when compared to adjacent non-tumor tissue. However, its diagnostic value and role in CRC have yet to be described. CXC chemokine receptor-4 (CXCR4), which participates in multiple cell processes such as cell invasion-related signaling pathways, was predicted to be a potential target of miR-133b. The aim of this study was to investigate the associations and functions of miR-133b and CXCR4 in CRC initiation and invasion. METHODS: Mature miR-133b and CXCR4 expression levels were detected in 31 tumor samples and their adjacent, non-tumor tissues from patients with CRC, as well as in 6 CRC cell lines, using real-time quantitative RT-PCR (qRT-PCR). Luciferase reporter assays and Western blots were used to validate CXCR4 as a putative target gene of miR-133b. Regulation of CXCR4 expression by miR-133b was assessed using qRT-PCR and Western blot analysis, and the effects of exogenous miR-133b and CXCR4 on cell invasion and migration were evaluated in vitro using the SW-480 and SW-620 CRC cell lines. RESULTS: A significant downregulation of miR-133b was observed in 93.55% of CRC tissues, and the expression of miR-133b was much lower in metastatic tumors (stage C and D, stratified by the Modified Dukes Staging System) than in primary tumors (stage A and B). In contrast, CXCR4 protein expression significantly increased in 52.63% of CRC samples, and increased CXCR4 expression in CRC was associated with advanced tumor stage. CXCR4 was shown to be a direct target of miR-133b by luciferase reporter assays, and transfection of miR-133b mimics inhibited invasion and stimulated apoptosis of SW-480 and SW-620 CRC cells. CONCLUSIONS: Our study demonstrated that downregulated miR-133b contributed to increased cell invasion and migration in CRC by negatively regulating CXCR4. These findings may be significant for the development of therapy target for CRC.

Diverse functions of miR-125 family in different cell contexts.

MicroRNAs (miRNAs) are emerging as a novel class of non-coding RNA molecules that regulate gene expression at a post-transcriptional level. More than 1000 miRNAs have been identified in human cells to date, and they are reported to play important roles in normal cell homeostasis, cell metastasis and disease pathogensis and progression. MiR-125, which is a highly conserved miRNA throughout diverse species from nematode to humans, consists of three homologs hsa-miR-125a, hsa-miR-125b-1 and hsa-miR-125-2. Members of this family have been validated to be down-regulated, exhibiting its disease-suppressing properties in many different types of diseases, while they also have disease-promoting functions in certain contexts. MiR-125 targets a number of genes such as transcription factors, matrix-metalloprotease, members of Bcl-2 family and others, aberrance of which may lead to abnormal proliferation, metastasis and invasion of cells, even carcinomas. Furthermore, miR-125 plays a crucial role in immunological host defense, especially in response to bacterial or viral infections. In this review, we summarize the implication of miR-125 family in disease suppression and promotion, focusing on carcinoma and host immune responses. We also discussed the potential of this miRNA family as promising biomarkers and therapeutic targets for different diseases in future.

Camptothecin induces apoptosis in cancer cells via microRNA-125b-mediated mitochondrial pathways.

Camptothecin (CPT) is an effective chemotherapeutic agent for treatment of patients with cancer. The mechanisms underlying CPT-mediated responses in cancer cells are not fully understood. MicroRNA (miRNA) play important roles in tumorigenesis and drug sensitivity. However, the interaction between camptothecin and miRNA has not been previously explored. In this study, we verified that miR-125b was down-regulated in CPT-induced apoptosis in cancer cells and that ectopic expression of miR-125b partially restored cell viability and inhibited cell apoptosis that was induced by CPT. In addition, we demonstrated that CPT induced apoptosis in cancer cells by miR-125b-mediated mitochondrial pathways via targeting to the 3'-untranslated (UTR) regions of Bak1, Mcl1, and p53. A significant increase in Bak1, Mcl1, and p53 protein levels was detected in response to the treatments of CPT. It is noteworthy that the expression levels of Bak1, Mcl1, and p53 increased in a time-dependent manner and negatively correlated with miR-125b expression. It is noteworthy that we revealed that miR-125b directly targeted the 3'UTR regions of multiple genes in a CPT-induced mitochondrial pathway. In addition, most targets of miR-125b were proapoptotic genes, whereas some of the targets were antiapoptotic genes. We hypothesized that miR-125b may mediate the activity of chemotherapeutic agents to induce apoptosis by regulating multiple targets. This is the first report to show that camptothecin induces cancer cell apoptosis via miRNA-mediated mitochondrial pathways. The results suggest that suppression of miR-125b may be a novel approach for the treatment of cancer.

miR-125b, a target of CDX2, regulates cell differentiation through repression of the core binding factor in hematopoietic malignancies.

MicroRNA-125b (miR-125b), a small noncoding RNA molecule, has been found to be deregulated and functions as an oncogene in many cancers including hematopoietic malignancies. However, the mechanisms accounting for miR-125b dysregulation remain to be elucidated. The present study aims to identify the factors that might contribute to up-regulation of miR-125b in human hematopoietic malignancies and its downstream targets for lineage-specific differentiation. We at first reported that CDX2, a homeobox transcription factor, binds to promoter regions of the miR-125b gene and activates transcriptional regulation of miR-125b in malignant myeloid cells. We further revealed that increasing levels of CDX2 in malignant myeloid cells activate miR-125b expression, which in turn inhibits core binding factor ß (CBFß) translation, thereby counteracting myeloid cell differentiation, at least for granulocytic lineage, and promoting leukemogenesis. Interestingly, we found that this novel pathway including CDX2, miR-125b, and CBFß was mediated by undergoing all-trans-retinoic acid induction. Once differentiation ensues with all-trans-retinoic acid treatment, CDX2 activity decreases, leading to a reduction in miR-125b transcription and up-regulation of CBFß in myeloid cells and in patients. The study provides a new mechanism that contributes to hematopoietic malignancies, which could involve deregulation of miR-125b and its up- and downstream factors. As altered expression of miRNAs has been reported in a wide range of malignancies, delineating the underlying molecular mechanisms of aberrant miRNA expression and characterizing the upstream and downstream factors will help to understand important steps in the pathogenesis of these afflictions.

A set of miRNAs that involve in the pathways of drug resistance and leukemic stem-cell differentiation is associated with the risk of relapse and glucocorticoid response in childhood ALL.

Relapse is a major challenge in the successful treatment of childhood acute lymphoblastic leukemia (ALL). Despite intensive research efforts, the mechanisms of ALL relapse are still not fully understood. An understanding of the molecular mechanisms underlying treatment outcome, therapy response and the biology of relapse is required. In this study, we carried out a genome-wide microRNA (miRNA) microarray analysis to determine the miRNA expression profiles and relapse-associated miRNA patterns in a panel of matched diagnosis-relapse or diagnosis-complete remission (CR) childhood ALL samples. A set of miRNAs differentially expressed either in relapsed patients or at diagnosis compared with CR was further validated by quantitative real-time polymerase chain reaction in an independent sample set. Analysis of the predicted functions of target genes based on gene ontology 'biological process' categories revealed that the abnormally expressed miRNAs are associated with oncogenesis, classical multidrug resistance pathways and leukemic stem cell self-renewal and differentiation pathways. Several targets of the miRNAs associated with ALL relapse were experimentally validated, including FOXO3, BMI1 and E2F1. We further investigated the association of these dysregulated miRNAs with clinical outcome and confirmed significant associations for miR-708, miR-223 and miR-27a with individual relapse-free survival. Notably, miR-708 was also found to be associated with the in vivo glucocorticoid therapy response and with disease risk stratification. These miRNAs and their targets might be used to optimize anti-leukemic therapy, and serve as novel targets for development of new countermeasures of leukemia. This fundamental study may also contribute to establish the mechanisms of relapse in other cancers.