MicroRNAs (miRNAs) with tumor-suppressor potential might have therapeutic applications in multiple myeloma (MM) through the modulation of still undiscovered molecular pathways. Here, we investigated the effects of enforced expression of miR-29b on the apoptotic occurrence in MM and highlighted its role in the context of a new transcriptional loop that is finely tuned by the proteasome inhibitor bortezomib. In details, in vitro growth inhibition and apoptosis of MM cells was induced by either transient expression of synthetic miR-29b or its stable lentivirus-enforced expression. We identified Sp1, a transcription factor endowed with oncogenic activity, as a negative regulator of miR-29b expression in MM cells. Since Sp1 expression and functions are regulated via the 26S proteasome, we investigated the effects of bortezomib on miR-29b-Sp1 loop, showing that miR-29b levels were indeed upregulated by the drug. At the same time, the bortezomib/miR-29b combination produced significant pro-apoptotic effects. We also demonstrated that the PI3K/AKT pathway plays a major role in the regulation of miR-29b-Sp1 loop and induction of apoptosis in MM cells. Finally, MM xenografts constitutively expressing miR-29b showed significant reduction of their tumorigenic potential. Our findings indicate that miR-29b is involved in a regulatory loop amenable of pharmacologic intervention and modulates the anti-MM activity of bortezomib in MM cells.
multiple myeloma; plasma cell leukemia; miR-29b; microRNA; miRNAs; Sp1; bortezomib
This article describes a novel role of Cdc25A down-regulation during differentiation of proliferating myoblasts.
Induction of a G1 phase cell cycle arrest, caused primarily by the inhibition of cyclin-dependent-kinase 2 (cdk2), is a critical step in the differentiation of myoblasts into myotubes. Here, we report that two microRNAs, miR-322/424 and miR-503, are induced and promote cdk2 inhibition during myogenesis. These microRNAs down-regulate Cdc25A, the phosphatase responsible for removing inhibitory phosphorylation of cdk2, both in myoblasts differentiating into myotubes and in nonmuscle cells. Cdc25A is down-regulated during muscle differentiation by multiple pathways: action of these two microRNAs, proteasomal degradation of Cdc25A protein and transcriptional repression. Overexpression of Cdc25A or of cdk2 with mutations on T14 and Y15 (cdk2-AF), so that it cannot be inhibited by phosphorylation, decreases differentiation and differentiation-induced cell cycle quiescence. Introduction of miR-322/424 and miR-503 in heterologous cancer cells induces G1 arrest, which is also attenuated by overexpression of the cdk2-AF mutant. Until now Cdc25A and the inhibitory phosphorylation on T14 and Y15 of cdk2 have only been implicated in the intra-S phase checkpoint pathway after DNA damage. Our results reveal an unexpected role of Cdc25A down-regulation and the inhibitory phosphorylation of cdk2 T14 and Y15 in cell cycle quiescence during muscle differentiation and implicate two muscle differentiation-induced microRNAs in the process.
MIR129-2 has been shown to be a tumor suppressor microRNA hypermethylated in epithelial cancers.
Patients and methods
Epigenetic inactivation of MIR129-2 was studied by methylation-specific PCR (MSP) in 13 cell lines (eight myeloma and five lymphoma), 15 normal controls and 344 primary samples including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), non-Hodgkin’s lymphoma (NHL), multiple myeloma (MM) at diagnosis, MM at relapse/progression, and monoclonal gammopathy of undetermined significance (MGUS). Expression of MIR129 and its target, SOX4, in cell lines was measured before and after hypomethylating treatment and MIR129 overexpression. MIR129 expression was correlated with MIR129-2 methylation status in primary lymphoma samples. Tumor suppressor function of MIR129 was demonstrated by MTT and trypan blue exclusion assay after MIR129 overexpression.
The sensitivity of the methylated-MSP was one in 103. Different MSP statuses, including complete methylation, partial methylation, and complete unmethylation, were verified by quantitative bisulfite pyrosequencing. All five lymphoma and seven of eight myeloma cell lines showed complete and partial MIR129-2 methylation. In primary samples, MIR129-2 methylation was absent in AML and CML, but detected in 5% ALL, 45.9% CLL, 49.5% MM at diagnosis, and 59.1% NHL. In CLL, MIR129-2 methylation adversely impacted on survival (p=0.004). In MM, MIR129-2 methylation increased from 27.5% MGUS to 49.5% MM at diagnosis and 41.5% at relapse/progression (p=0.023). In NHL, MIR129-2 methylation was associated with MIR124-1 and MIR203 methylation (p<0.001), and lower MIR129 expression (p=0.009). Hypomethylation treatment of JEKO-1, homozygously methylated for MIR129-2, led to MIR129-2 demethylation and MIR129 re-expression, with downregulation of SOX4 mRNA. Moreover, MIR129 overexpression in both mantle cell lines, JEKO-1 and GRANTA-519, inhibited cellular proliferation and enhanced cell death, with concomitant SOX4 mRNA downregulation.
MIR129-2 is a tumor suppressive microRNA frequently methylated in lymphoid but not myeloid malignancies, leading to reversible MIR129-2 silencing. In CLL, MIR129-2 methylation was associated with an inferior survival. In MM, MIR129-2 methylation might be acquired during progression from MGUS to symptomatic MM. In NHL, MIR129-2 methylation might collaborate with MIR124-1 and MIR203 methylation in lymphomagenesis.
microRNA; Tumor suppressor; Hypermethylation; MIR129; Hematological cancers
The proteasome inhibitor bortezomib is clinically approved for the treatment of multiple myeloma. However, long-term remissions are difficult to achieve, and myeloma cells often develop secondary resistance to proteasome inhibitors. We recently demonstrated that the extraordinary sensitivity of myeloma cells toward bortezomib is dependent on their extensive immunoglobulin synthesis, thereby triggering the terminal unfolded protein response (UPR). Here, we investigated whether verapamil, an inhibitor of the multidrug resistance (MDR) gene product, can enhance the cytotoxicity of bortezomib. The combination of bortezomib and verapamil synergistically decreased the viability of myeloma cells by inducing cell death. Importantly, bortezomib-mediated activation of major UPR components was enhanced by verapamil. The combination of bortezomib and verapamil resulted in caspase activation followed by poly(ADP-ribose) polymerase cleavage, whereas nuclear factor κB (NF-κB) activity declined in myeloma cells. Also, we found reduced immunoglobulin G secretion along with increased amounts of ubiquitinylated proteins within insoluble fractions of myeloma cells when using the combination treatment. Verapamil markedly induced reactive oxygen species production and autophagic-like processes. Furthermore, verapamil decreased MDR1 expression. We conclude that verapamil increased the antimyeloma effect of bortezomib by enhancing ER stress signals along with NF-κB inhibition, leading to cell death. Thus, the combination of bortezomib with verapamil may improve the efficacy of proteasome inhibitory therapy.
The findings presented indicate that polyamine-regulated CUG-binding protein 1 and microRNA-222 modulate cyclin-dependent kinase 4 (CDK4) translation at least in part by altering the recruitment of CDK4 mRNA to processing bodies.
The amino acid–derived polyamines are organic cations that are essential for growth in all mammalian cells, but their exact roles at the molecular level remain largely unknown. Here we provide evidence that polyamines promote the translation of cyclin-dependent kinase 4 (CDK4) by the action of CUG-binding protein 1 (CUGBP1) and microRNA-222 (miR-222) in intestinal epithelial cells. Both CUGBP1 and miR-222 were found to bind the CDK4 mRNA coding region and 3′-untranslated region and repressed CDK4 translation synergistically. Depletion of cellular polyamines increased cytoplasmic CUGBP1 abundance and miR-222 levels, induced their associations with the CDK4 mRNA, and inhibited CDK4 translation, whereas increasing the levels of cellular polyamines decreased CDK4 mRNA interaction with CUGBP1 and miR-222, in turn inducing CDK4 expression. Polyamine-deficient cells exhibited an increased colocalization of tagged CDK4 mRNA with processing bodies; this colocalization was abolished by silencing CUGBP1 and miR-222. Together, our findings indicate that polyamine-regulated CUGBP1 and miR-222 modulate CDK4 translation at least in part by altering the recruitment of CDK4 mRNA to processing bodies.
miR-124-1 is a tumour suppressor microRNA (miR). Epigenetic deregulation of miRs is implicated in carcinogenesis. Promoter DNA methylation and histone modification of miR-124-1 was studied in 5 normal marrow controls, 4 lymphoma, 8 multiple myeloma (MM) cell lines, 230 diagnostic primary samples of acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myeloid leukaemia (CML), chronic lymphocytic leukaemia (CLL), MM, and non-Hodgkin's lymphoma (NHL), and 53 MM samples at stable disease or relapse. Promoter of miR-124-1 was unmethylated in normal controls but homozygously methylated in 4 of 4 lymphoma and 4 of 8 myeloma cell lines. Treatment of 5-Aza-2′-deoxycytidine led to miR-124-1 demethylation and re-expression of mature miR-124, which also associated with emergence of euchromatic trimethyl H3K4 and consequent downregulation of CDK6 in myeloma cells harboring homozygous miR-124-1 methylation. In primary samples at diagnosis, miR-124-1 methylation was absent in CML but detected in 2% each of MM at diagnosis and relapse/progression, 5% ALL, 15% AML, 14% CLL and 58.1% of NHL (p<0.001). Amongst lymphoid malignancies, miR-124-1 was preferentially methylated in NHL than MM, CLL or ALL. In primary lymphoma samples, miR-124-1 was preferentially hypermethylated in B- or NK/T-cell lymphomas and associated with reduced miR-124 expression. In conclusion, miR-124-1 was hypermethylated in a tumour-specific manner, with a heterochromatic histone configuration. Hypomethylation led to partial restoration of euchromatic histone code and miR re-expression. Infrequent miR-124-1 methylation detected in diagnostic and relapse MM samples showed an unimportant role in MM pathogenesis, despite frequent methylation found in cell lines. Amongst haematological cancers, miR-124-1 was more frequently hypermethylated in NHL, and hence warrants further study.
Pulmonary hypertension (PH) is driven by diverse pathogenic etiologies. Owing to their pleiotropic actions, microRNA (miRNA) are potential candidates for coordinated regulation of these disease stimuli.
Methods and Results
Using a network biology approach, we identify miRNA associated with multiple pathogenic pathways central to PH. Specifically, microRNA-21 (miR-21) is predicted as a PH-modifying miRNA, regulating targets integral to bone morphogenetic protein (BMP) and Rho/Rho kinase signaling as well as functional pathways associated with hypoxia, inflammation, and genetic haplo insufficiency of the BMP Receptor Type 2 (BMPRII). To validate these predictions, we have found that hypoxia and BMPRII signaling independently up-regulate miR-21 in cultured pulmonary arterial endothelial cells. In a reciprocal feedback loop, miR-21 down-regulates BMPRII expression. Furthermore, miR-21 directly represses RhoB expression and Rho kinase activity, inducing molecular changes consistent with decreased angiogenesis and vasodilation. In vivo, miR-21 is up-regulated in pulmonary tissue from several rodent models of PH and in humans with PH. Upon induction of disease in miR-21-null mice, RhoB expression and Rho-kinase activity are increased, accompanied by exaggerated manifestations of PH.
A network-based bioinformatic approach coupled with confirmatory in vivo data delineates a central regulatory role for miR-21 in PH. Furthermore, this study highlights the unique utility of network biology for identifying disease-modifying miRNA in PH.
Pulmonary Heart Disease; microRNA; Network Biology; Molecular Biology; Vasculature
Two key features of myeloma cells are the deregulation of the cell cycle and the dependency on the expression of the BCL2 family of anti-apoptotic proteins. The cell division cycle 7 (CDC7) is an essential S-phase kinase and emerging CDC7 inhibitors are effective in a variety of preclinical cancer models. These compounds also inhibit CDK9 which is relevant for MCL-1 expression. The activity and mechanism of action of the dual CDC7/CDK9 inhibitor PHA-767491 was assessed in a panel of multiple myeloma cell lines, in primary samples from patients, in the presence of stromal cells and in combination with drugs used in current chemotherapeutic regimens. We report that in all conditions myeloma cells undergo cell death upon PHA-767491 treatment and we report an overall additive effect with melphalan, bortezomib and doxorubicin, thus supporting further assessment of targeting CDC7 and CDK9 in multiple myeloma.
apoptosis; BCL2-family; DNA replication; multiple myeloma; kinase inhibitor; cell cycle
BM mesenchymal stromal cells (BM-MSCs) support multiple myeloma (MM) cell growth, but little is known about the putative mechanisms by which the BM microenvironment plays an oncogenic role in this disease. Cell-cell communication is mediated by exosomes. In this study, we showed that MM BM-MSCs release exosomes that are transferred to MM cells, thereby resulting in modulation of tumor growth in vivo. Exosomal microRNA (miR) content differed between MM and normal BM-MSCs, with a lower content of the tumor suppressor miR-15a. In addition, MM BM-MSC–derived exosomes had higher levels of oncogenic proteins, cytokines, and adhesion molecules compared with exosomes from the cells of origin. Importantly, whereas MM BM-MSC–derived exosomes promoted MM tumor growth, normal BM-MSC exosomes inhibited the growth of MM cells. In summary, these in vitro and in vivo studies demonstrated that exosome transfer from BM-MSCs to clonal plasma cells represents a previously undescribed and unique mechanism that highlights the contribution of BM-MSCs to MM disease progression.
A rising body of evidence suggests that silencing microRNAs (miRNAs) with oncogenic potential may represent a successful therapeutic strategy for human cancer. We investigated the therapeutic activity of miR-221/222 inhibitors against human multiple myeloma (MM) cells. Enforced expression of miR-221/222 inhibitors triggered in vitro anti-proliferative effects and up-regulation of canonic miR-221/222 targets, including p27Kip1, PUMA, PTEN and p57Kip2, in MM cells highly expressing miR-221/222. Conversely, transfection of miR-221/222 mimics increased S-phase and down-regulated p27Kip1 protein expression in MM with low basal miR-221/222 levels. The effects of miR-221/222 inhibitors was also evaluated in MM xenografts in SCID/NOD mice. Significant anti-tumor activity was achieved in xenografted mice by the treatment with miR-221/222 inhibitors, together with up-regulation of canonic protein targets in tumors retrieved from animals. These findings provide proof of principle that silencing the miR-221/222 cluster exerts significant therapeutic activity in MM cells with high miR-221/222 level of expression, which mostly occurs in TC2 and TC4 MM groups. These findings suggest that MM genotyping may predict the therapeutic response. All together our results support a framework for clinical development of miR-221/222 inhibitors-based therapeutic strategy in this still incurable disease.
miR-221; miR-222; microRNA; miRNA; multiple myeloma; plasma cell leukemia
Chromatin remodeling factors are becoming known as crucial facilitators of recruitment of repair proteins to sites of DNA damage. Multiple chromatin remodeling protein complexes are now known to be required for efficient double strand break repair. In a screen for microRNAs that modulate the DNA damage response, we discovered that expression of the miR-99 family of microRNAs correlates with radiation sensitivity. These microRNAs were also transiently induced following radiation. The microRNAs target the SWI/SNF chromatin remodeling factor SNF2H/SMARCA5, a component of the ACF1 complex. We found that by reducing levels of SNF2H, miR-99a reduced BRCA1 localization to sites of DNA damage. Introduction of the miR-99 family of microRNAs into cells reduced the rate and overall efficiency of repair by both homologous recombination and non-homologous end joining. Finally, induction of the miR-99 family following radiation prevents an increase in SNF2H expression and reduces the recruitment of BRCA1 to sites of DNA damage following a second dose of radiation, reducing the efficiency of repair after multiple rounds of radiation as used in fractionated radiotherapy.
microRNA; DNA repair; cancer; SNF2H; miR-99
CK2 is a pivotal pro-survival protein kinase in multiple myeloma that may likely impinge on bortezomib-regulated cellular pathways. In the present study, we investigated CK2 expression in multiple myeloma and mantle cell lymphoma, two bortezomib-responsive B cell tumors, as well as its involvement in bortezomib-induced cytotoxicity and signaling cascades potentially mediating bortezomib resistance. In both tumors, CK2 expression correlated with that of its activated targets NF-κB and STAT3 transcription factors. Bortezomib-induced proliferation arrest and apoptosis were significantly amplified by the simultaneous inhibition of CK2 with two inhibitors (CX-4945 and K27) in multiple myeloma and mantle cell lymphoma cell lines, in a model of multiple myeloma bone marrow microenvironment and in cells isolated from patients. CK2 inhibition empowered bortezomib-triggered mitochondrial-dependent cell death. Phosphorylation of NF-κB p65 on Ser529 (a CK2 target site) and rise of the levels of the endoplasmic reticulum stress kinase/endoribonuclease Ire1α were markedly reduced upon CK2 inhibition, as were STAT3 phospho Ser727 levels. On the contrary, CK2 inhibition increased phospho Ser51 eIF2α levels and enhanced the bortezomib-dependent accumulation of poly-ubiquitylated proteins and of the proteotoxic stress-associated chaperone Hsp70. Our data suggest that CK2 over expression in multiple myeloma and mantle cell lymphoma cells might sustain survival signaling cascades and can antagonize bortezomib-induced apoptosis at different levels. CK2 inhibitors could be useful in bortezomib-based combination therapies.
Although the immune response is predominantly controlled at the transcriptional level, microRNA-mediated RNA interference is emerging as an important regulatory mechanism that operates at the translation level. Specifically, recent studies indicate that those miRNAs that are selectively and/or highly expressed in immune cells including the miR-17–92 cluster, miR-150, miR-155, miR-181 and miR-223 have a ‘permissive’ function in the maturation, proliferation and differentiation of myeloid and lymphoid cells. Importantly, these actions of miRNAs often involve interactions with transcription factors. In contrast, the rapid and transient induction of miR-9, miR-146a and miR-155 has been speculated to negatively regulate the acute responses following activation of innate immune through down-regulation of proteins involved in the receptor-induced signalling pathways.
Recent studies have demonstrated that one-third of known microRNAs (miRNAs) are stably detectable in plasma. Therefore, we assessed plasma miRNAs to investigate the dynamics of oncomir 17-92a, which is highly expressed in multiple myeloma (MM) patients. The plasma miR-92a level in symptomatic MM patients was significantly downregulated compared with normal subjects (P<0.0001), regardless of immunoglobulin subtypes or disease stage at diagnosis. In contrast, miR-92a levels in peripheral blood CD8+ or CD4+ cells from MM patients were lower than those of normal subjects, and the miR-92a levels of the cells tended to correlate with plasma miR-92a levels. The plasma miR-92a level in the complete remission group became normalized, whereas the partial response (PR) and very good PR groups did not reach the normal range. In smoldering MM, the plasma miR-92a level did not show a significant difference compared with normal subjects. Our findings suggest that measurement of the plasma miR-92a level in MM patients could be useful for initiation of chemotherapy and monitoring disease status, and the level may represent, in part, the T-cell immunity status of these patients.
multiple myeloma; plasma miR-92a; T lymphocytes
MicroRNAs are well-suited to regulate tumor metastasis due to their capacity to coordinately repress numerous target genes, thereby potentially enabling their intervention at multiple steps of the invasion-metastasis cascade. We identify a microRNA exemplifying these attributes, miR-31, whose expression correlates inversely with metastasis in human breast cancer patients. Overexpression of miR-31 in otherwise-aggressive breast tumor cells suppresses metastasis. We deploy a stable microRNA sponge strategy to stably inhibit miR-31 in vivo; this allows otherwise-non-aggressive breast cancer cells to metastasize. These phenotypes do not involve confounding influences on primary tumor development and are specifically attributable to miR-31-mediated inhibition of several steps of metastasis, including local invasion, extravasation or initial survival at a distant site, and metastatic colonization. Such pleiotropy is achieved via coordinate repression of a cohort of metastasis-promoting genes, including RhoA. Indeed, RhoA re-expression partially reverses miR-31-imposed metastasis-suppression. These findings indicate that miR-31 uses multiple mechanisms to oppose metastasis.
(−)-Epigallocatechin-3-gallate (EGCG) has been reported to affect many cellular regulatory pathways. This study aims to determine whether EGCG could target microRNA (miRNA), one of the mechanisms for cells to achieve subtle change in multiple targets. We found that, in both human and mouse lung cancer cells in culture, EGCG specifically upregulated the expression of miR-210, a major miRNA regulated by HIF-1α. Furthermore, we found that overexpression of miR-210 led to reduced cell proliferation rate and anchorage-independent growth as well as reduced sensitivity to EGCG. On the mechanisms of miR-210 regulation by EGCG, we demonstrated that the regulation was mediated through the hypoxia-response element in miR-210 promoter. Consistently, the upregulation of miR-210 was found to be correlated with the stabilized HIF-1α in lung cancer cell lines after EGCG treatment. This EGCG-induced stabilization of HIF-1α was further shown by the stabilization of HA-tagged HIF-1α but not the P402A/P564A-mutated HIF-1α by EGCG, suggesting that EGCG targets the oxygen-dependent degradation (ODD) domain. Direct evidence was obtained by affinity binding assay showing that EGCG specifically binds HIF-1α with a Kd = 3.47 μM. This result suggests that EGCG binding interferes with the hydroxylation of key Pro residues in the ODD domain, preventing HIF-1α from the Pro hydroxylation-dependent ubiquitination and subsequent proteosome-mediated degradation. In summary, our results demonstrated, for the first time, the elevation of miR-210 by EGCG in lung cancer cell lines and this is mediated by the stabilization of HIF-1α. This event contributes to the anticancer activity of EGCG.
In response to DNA damage, cells activate a complex, kinase-based signaling network to arrest the cell cycle and allow time for DNA repair, or, if the extend of damage is beyond repair capacity, induce apoptosis. This signaling network, which is collectively referred to as the DNA damage response (DDR), is primarily thought to consist of two components—a rapid phosphorylation-driven signaling cascade that results in immediate inhibition of Cdk/cyclin complexes and a delayed transcriptional response that promotes a prolonged cell cycle arrest through the induction of Cdk inhibitors, such as p21. In recent years a third layer of complexity has emerged that involves potent posttranscriptional regulatory mechanisms that control the cellular response to DNA damage. Although much has been written on the relevance of the DDR in cancer and on the post-transcriptional role of microRNAs (miRs) in cancer, the post-transcriptional regulation of the DDR by non-coding RNAs and RNA-binding proteins (RBPs) still remains elusive in large parts. Here, we review the recent developments in this exciting new area of research in the cellular response to genotoxic stress. We put specific emphasis on the role of RBPs and the control of their function through DNA damage-activated protein kinases.
MAPKAP-kinase 2; HuR; hnRNP A0; TIAR; PARN; DNA damage response; cell cycle checkpoint
Androgen Receptor (AR) mediated oncogenic pathways have not been fully elucidated. In this study we utilized high throughput microarray analysis on two AR-positive Prostate Cancer (CaP) cell lines to identify 16 AR-responsive microRNAs (miRNAs). We focused on miR-21 because of its previously reported oncogenic activity in other cancers. We demonstrate androgen-induced AR binding to the defined miR-21 promoter, miPPR-21, suggesting direct transcriptional regulation. Moreover, inhibition of miR-21 diminished androgen-induced CaP cell proliferation, providing new evidence that microRNAs can contribute to androgen-driven cell growth. Elevated expression of miR-21 enhanced CaP tumor growth in vivo and, surprisingly, was sufficient for androgen-dependent tumors to overcome castration-mediated growth arrest. Thus, elevated miR-21 expression alone is sufficient to impart castration resistance. Moreover, quantitative RT-PCR analysis revealed elevated miR-21 expression in CaP when compared to adjacent normal tissue. These results suggest that miR-21 may contribute to CaP pathogenesis.
Prostate cancer; microRNA; Androgen Receptor
Bortezomib is a specific inhibitor of proteasomes, the most important protease complexes in protein degradation. Bortezomib can induce apoptosis of a variety of cancer cells, including leukemia, lymphoma, multiple myeloma, breast cancers, prostate cancers, lung cancers, and so on. However, extensive studies and overall evaluation suggested that multiple myeloma is the most sensitive and the best responsive disease which was later approved by Food and Drug Administration for bortezomib treatment. Because proteasomes are an essential component in the ubiquitin-proteasomal protein degradation pathway, the discovery of bortezomib implicates that the UPS is critical for myeloma pathophysiology. The UPS also contains ubiquitin, ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), ubiquitin ligases (E3) and deubiquitinases (Dubs). In this review, we examined and analyzed the recent advancements of the UPS components in multiple myeloma and its implications in drug discovery for myeloma treatment.
Ubiquitin-proteasomal system; deubiquitinases; bortezomib; multiple myeloma; drug discovery
MicroRNAs regulated by LPS target genes that contribute to the inflammatory phenotype. Here we show that Akt1, which is activated by LPS, differentially regulates miRNAs including let-7e, miR-155, miR-181c and miR125b. In silico analyses and transfection studies revealed that let-7e represses TLR4 while miR-155 represses SOCS1, two genes critical for LPS-driven TLR signalling, which regulate endotoxin sensitivity and tolerance. As a result, Akt1−/− macrophages exhibited increased responsiveness to LPS in culture and Akt1−/− mice did not develop endotoxin tolerance in vivo. Overexpression of let-7e and suppression of miR-155 in Akt1−/− macrophages restores sensitivity and tolerance to LPS in culture and in animals. These results indicate that Akt1 regulates the response of macrophages to LPS by controlling miRNA expression.
Akt; miRNA; Toll-like receptor 4; LPS; SOCS1
Tolerance represents a critical component of addiction. The large conductance calcium-and voltage-activated potassium channel (BK) is a well-established alcohol target, and an important element in behavioral and molecular alcohol tolerance. We tested whether microRNA, a newly-discovered class of gene expression regulators, plays a role in the development of tolerance. We show that in adult mammalian brain alcohol upregulates microRNA (miR-9) and mediates post-transcriptional reorganization in BK mRNA splice variants by miR-9-dependent destabilization of BK mRNAs containing 3’UTRs with a miR-9 Recognition Element (MRE). Different splice variants encode BK isoforms with different alcohol sensitivities. Computational modeling indicates that this miR-9 dependent mechanism contributes to alcohol tolerance. Moreover, this mechanism can be extended to regulation of additional miR-9 targets relevant to alcohol abuse. Our results describe a novel mechanism of multiplex regulation of stability of alternatively spliced mRNA by miRNA in drug adaptation and neuronal plasticity.
The microRNA miR-22 targets CDK6, SIRT1, and Sp1—genes involved in regulation of the senescence program—to suppress cell growth and proliferation.
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.
Advanced cutaneous T-cell lymphoma (CTCL) is resistant to chemotherapy and presents a major area of medical need. In view of the known role of microRNAs (miRNAs) in the regulation of cellular signalling, we aimed to identify the functionally important miRNA species, which regulate apoptosis in CTCL. Using a recently established model in which apoptosis of CTCL cell lines is induced by Notch-1 inhibition by γ-secretase inhibitors (GSIs), we found that miR-122 was significantly increased in the apoptotic cells. miR-122 up-regulation was not specific for GSI-1 but was also seen during apoptosis induced by chemotherapies including doxorubicin and proteasome blockers (bortezomib, MG132). miR-122 was not expressed in quiescent T-cells, but was detectable in CTCL: in lesional skin in mycosis fungoides and in Sézary cells purified from peripheral blood. In situ hybridization results showed that miR-122 was expressed in the malignant T-cell infiltrate and increased in the advanced stage mycosis fungoides. Surprisingly, miR-122 overexpression decreased the sensitivity to the chemotherapy-induced apoptosis via a signaling circuit involving the activation of Akt and inhibition of p53. We have also shown that induction of miR-122 occurred via p53 and that p53 post-transcriptionally up-regulated miR-122. miR-122 is thus an amplifier of the antiapoptotic Akt/p53 circuit and it is conceivable that a pharmacological intervention in this pathway may provide basis for novel therapies for CTCL.
Aberrant DNA methylation plays a relevant role in multiple myeloma (MM) pathogenesis. MicroRNAs
(miRNAs) are a class of small non-coding RNAs that recently emerged as master regulator of gene
expression by targeting protein-coding mRNAs. However, miRNAs involvement in the regulation of the
epigenetic machinery and their potential use as therapeutics in MM remain to be investigated. Here,
we provide evidence that the expression of de novo DNA methyltransferases (DNMTs) is deregulated in
MM cells. Moreover, we show that miR-29b targets DNMT3A and DNMT3B mRNAs and reduces global DNA
methylation in MM cells. In vitro transfection of MM cells with synthetic miR-29b mimics
significantly impairs cell cycle progression and also potentiates the growth-inhibitory effects
induced by the demethylating agent 5-azacitidine. Most importantly, in vivo intratumor or systemic
delivery of synthetic miR-29b mimics, in two clinically relevant murine models of human MM,
including the SCID-synth-hu system, induces significant anti-tumor effects. All together, our
findings demonstrate that aberrant DNMTs expression is efficiently modulated by tumor suppressive
synthetic miR-29b mimics, indicating that methyloma modulation is a novel matter of investigation in
miRNA-based therapy of MM.
miR-29b; microRNA; multiple myeloma; DNA methyltransferases; DNMT
MicroRNA-29b (miR-29b) expression has been shown to be reduced in non-small–cell lung cancer (NSCLC) tissues. Here, we have identified the oncogene cyclin-dependent protein kinase 6 (CDK6) as a direct target of miR-29b in lung cancer. We hypothesized that in vivo restoration of miR-29b and thus targeting of genes important to tumor initiation and progression may represent an option for lung cancer treatment. We developed a cationic lipoplexes (LPs)-based carrier that efficiently delivered miR-29b both in vitro and in vivo. LPs containing miR-29b (LP-miR-29b) efficiently delivered miR-29b to NSCLC A549 cells, reduced the expression of key targets CDK6, DNMT3B, and myeloid cell leukemia sequence 1 (MCL1), as well as cell growth and clonogenicity of A549 cells. In addition, the IC50 for cisplatin in the miR-29b–treated cells was effectively reduced. In a xenograft murine model, LPs efficiently accumulated at tumor sites. Systemic delivery of LP-miR-29b increased the tumor miR-29b expression by approximately fivefold, downregulated the tumor mRNA expression of CDK6, DNMT3B, and MCL1 by ~57.4, ~40.5, and ~52.4%, respectively, and significantly inhibited tumor growth by ~60% compared with LP-miR-NC (negative control). Our results demonstrate that cationic LPs represent an efficient delivery system that holds great potential in the development of miRNA-based therapeutics for lung cancer treatment.
cationic lipoplexes; lung cancer; microRNA