A pan-cancer atlas of somatic mutations in miRNA biogenesis genes.

It is a well-known and intensively studied phenomenon that the levels of many miRNAs are differentiated in cancer. miRNA biogenesis and functional expression are complex processes orchestrated by many proteins cumulatively called miRNA biogenesis proteins. To characterize cancer somatic mutations in the miRNA biogenesis genes and investigate their potential impact on the levels of miRNAs, we analyzed whole-exome sequencing datasets of over 10 000 cancer/normal sample pairs deposited within the TCGA repository. We identified and characterized over 3600 somatic mutations in 29 miRNA biogenesis genes and showed that some of the genes are overmutated in specific cancers and/or have recurrent hotspot mutations (e.g. SMAD4 in PAAD, COAD and READ; DICER1 in UCEC; PRKRA in OV and LIN28B in SKCM). We identified a list of miRNAs whose level is affected by particular types of mutations in either SMAD4, SMAD2 or DICER1 and showed that hotspot mutations in the RNase domains in DICER1 not only decrease the level of 5p-miRNAs but also increase the level of 3p-miRNAs, including many well-known cancer-related miRNAs. We also showed an association of the mutations with patient survival. Eventually, we created an atlas/compendium of miRNA biogenesis alterations providing a useful resource for different aspects of biomedical research.

Mutational and phenotypic characterization of hereditary hemorrhagic telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular dysplasia. Care delivery for HHT patients is impeded by the need for laborious, repeated phenotyping and gaps in knowledge regarding the relationships between causal DNA variants in ENG, ACVRL1, SMAD4 and GDF2, and clinical manifestations. To address this, we analyzed DNA samples from 183 previously uncharacterized, unrelated HHT and suspected HHT cases using the ThromboGenomics high-throughput sequencing platform. We identified 127 rare variants across 168 heterozygous genotypes. Applying modified American College of Medical Genetics and Genomics Guidelines, 106 variants were classified as pathogenic/likely pathogenic and 21 as nonpathogenic (variant of uncertain significance/benign). Unlike the protein products of ACVRL1 and SMAD4, the extracellular ENG amino acids are not strongly conserved. Our inferences of the functional consequences of causal variants in ENG were therefore informed by the crystal structure of endoglin. We then compared the accuracy of predictions of the causal gene blinded to the genetic data using 2 approaches: subjective clinical predictions and statistical predictions based on 8 Human Phenotype Ontology terms. Both approaches had some predictive power, but they were insufficiently accurate to be used clinically, without genetic testing. The distributions of red cell indices differed by causal gene but not sufficiently for clinical use in isolation from genetic data. We conclude that parallel sequencing of the 4 known HHT genes, multidisciplinary team review of variant calls in the context of detailed clinical information, and statistical and structural modeling improve the prognostication and treatment of HHT.

Structural based screening of potential inhibitors of SMAD4: a step towards personalized medicine for gall bladder and other associated cancers.

Gall bladder cancer (GBC) is an aggressive and most common malignancy of biliary tract lacking effective treatment due to unavailability of suitable biomarkers and therapeutics. SMAD4 is an essential mediator of transforming growth factor-ß pathway involved in various cellular processes like growth, differentiation and apoptosis and also recognized as therapeutic target for GBC and other gastrointestinal tract cancers. In the present study, 3D structure of SMAD4 mutants was optimized through molecular dynamics simulation (MDS) along with wildtype. Furthermore, binding site of protein was predicted through hybrid approach and structural based virtual screening against two drug libraries was performed followed by docking. MDS of top docking score protein-ligand complexes were carried, and binding free energy was rescored. Two potential inhibitors, namely ZINC2098840 and ZINC8789167, were screened that displayed higher binding affinity towards mutant proteins compared with wildtype and both hydrophilic as well as hydrophobic interactions play a crucial role during protein-ligand binding. Current study identified novel and potent inhibitors of SMAD4 mutant that could be used as a drug candidate for the development of personalized medicine for gall bladder and other associated cancers.

A comparative analysis of Smad-responsive motifs identifies multiple regulatory inputs for TGF-ß transcriptional activation.

Smad proteins are transcriptional regulators activated by TGF-ß. They are known to bind to two distinct Smad-responsive motifs, namely the Smad-binding element (SBE) (5'-GTCTAGAC-3') and CAGA motifs (5'-AGCCAGACA-3' or 5'-TGTCTGGCT-3'). However, the mechanisms by which these motifs promote Smad activity are not fully elucidated. In this study, we performed DNA CASTing, binding assays, ChIP sequencing, and quantitative RT-PCR to dissect the details of Smad binding and function of the SBE and CAGA motifs. We observed a preference for Smad3 to bind CAGA motifs and Smad4 to bind SBE, and that either one SBE or a triple-CAGA motif forms a cis-acting functional half-unit for Smad-dependent transcription activation; combining two half-units allows efficient activation. Unexpectedly, the extent of Smad binding did not directly correlate with the abilities of Smad-binding sequences to induce gene expression. We found that Smad proteins are more tolerant of single bp mutations in the context of the CAGA motifs, with any mutation in the SBE disrupting function. CAGA and CAGA-like motifs but not SBE are widely distributed among stimulus-dependent Smad2/3-binding sites in normal murine mammary gland epithelial cells, and the number of CAGA and CAGA-like motifs correlates with fold-induction of target gene expression by TGF-ß. These data, demonstrating Smad responsiveness can be tuned by both sequence and number of repeats, provide a compelling explanation for why CAGA motifs are predominantly used for Smad-dependent transcription activation in vivo.

TGIF1 homeodomain interacts with Smad MH1 domain and represses TGF-ß signaling.

TGIF1 is a multifunctional protein that represses TGF-ß-activated transcription by interacting with Smad2-Smad4 complexes. We found that the complex structure of TGIF1-HD bound to the TGACA motif revealed a combined binding mode that involves the HD core and the major groove, on the one hand, and the amino-terminal (N-term) arm and the minor groove of the DNA, on the other. We also show that TGIF1-HD interacts with the MH1 domain of Smad proteins, thereby indicating that TGIF1-HD is also a protein-binding domain. Moreover, the formation of the HD-MH1 complex partially hinders the DNA-binding site of the complex, preventing the efficient interaction of TGIF1-HD with DNA. We propose that the binding of the TGIF1 C-term to the Smad2-MH2 domain brings both the HD and MH1 domain into close proximity. This local proximity facilitates the interaction of these DNA-binding domains, thus strengthening the formation of the protein complex versus DNA binding. Once the protein complex has been formed, the TGIF1-Smad system would be released from promoters/enhancers, thereby illustrating one of the mechanisms used by TGIF1 to exert its function as an active repressor of Smad-induced TGF-ß signaling.

A peptide that blocks the interaction of NF-κB p65 subunit with Smad4 enhances BMP2-induced osteogenesis.

Bone morphogenetic protein (BMP) potentiates bone formation through the Smad signaling pathway in vitro and in vivo. The transcription factor nuclear factor κB (NF-κB) suppresses BMP-induced osteoblast differentiation. Recently, we identified that the transactivation (TA) 2 domain of p65, a main subunit of NF-κB, interacts with the mad homology (MH) 1 domain of Smad4 to inhibit BMP signaling. Therefore, we further attempted to identify the interacting regions of these two molecules at the amino acid level. We identified a region that we term the Smad4-binding domain (SBD), an amino-terminal region of TA2 that associates with the MH1 domain of Smad4. Cell-permeable SBD peptide blocked the association of p65 with Smad4 and enhanced BMP2-induced osteoblast differentiation and mineralization without affecting the phosphorylation of Smad1/5 or the activation of NF-κB signaling. SBD peptide enhanced the binding of the BMP2-inudced phosphorylated Smad1/5 on the promoter region of inhibitor of DNA binding 1 (Id-1) compared with control peptide. Although SBD peptide did not affect BMP2-induced chondrogenesis during ectopic bone formation, the peptide enhanced BMP2-induced ectopic bone formation in subcortical bone. Thus, the SBD peptide is useful for enabling BMP2-induced bone regeneration without inhibiting NF-κB activity.

Sumoylation of SMAD 4 ameliorates the oxidative stress-induced apoptosis in osteoblasts.

Oxidative stress-induced mitochondrial function and cell apoptosis to osteoblasts, plays a critical role in the pathophysiology of osteoporosis. However, mechanisms underlying such process remain not yet clear. We aims in this study to investigate a possible role of SMAD (the mothers against decapentaplegic homolog 4 (SMAD4) in the oxidative stress-induced apoptosis, in homo sapiens osteoblast hFOB1.19 cells. Results demonstrated that the treatment with more than 100µM H2O2 significantly downregulated the cellular viability, whereas markedly induced apoptosis in hFOB1.19 cells. The SMAD4 was markedly reduced in both mRNA and protein levels in the H2O2 -treated hFOB1.19 cells, along with the reduction of Small ubiquitin-related modifier 1 (SUMO 1) and SUMO 2/3. The immunoprecipitation assay confirmed indicated the interaction between SUMO 1 (or SUMO 2/3) and SMAD4. Moreover, the SMAD4 overexpression markedly ameliorated the H2O2-resulted viability reduction and apoptosis induction in hFOB1.19 cells. Interestingly, such amelioration was blocked by the knockdown of SUMO 2/3. Taken together, we conclued that SMAD4 inhibits the H2O2-induced apoptosis in osteoblast hFOB1.19 cells; such inhibition might depend on the SUMOylation by SUMO 2/3. It implies a promising role of SMAD4 in oxidative stress-promoted damage to osteoblasts.

Myhre syndrome.

Myhre syndrome (MS) is a developmental disorder characterized by typical facial dysmorphism, thickened skin, joint limitation and muscular pseudohypertrophy. Other features include brachydactyly, short stature, intellectual deficiency with behavioral problems and deafness. We identified SMAD4 as the gene responsible for MS. The identification of SMAD4 mutations in Laryngotracheal stenosis, Arthropathy, Prognathism and Short stature (LAPS) cases supports that LAPS and MS are a unique entity. The long-term follow up of patients shows that these conditions are progressive with life threatening complications. All mutations are de novo and changing in the majority of cases Ile500, located in the MH2 domain involved in transcriptional activation. We further showed an impairment of the transcriptional regulation via TGFß target genes in patient fibroblasts. Finally, the absence of SMAD4 mutations in three MS cases may support genetic heterogeneity.

Novel SMAD4 mutation causing Myhre syndrome.

Myhre syndrome (MYHRS, OMIM 139210) is an autosomal dominant disorder characterized by developmental and growth delay, athletic muscular built, variable cognitive deficits, skeletal anomalies, stiffness of joints, distinctive facial gestalt and deafness. Recently, SMAD4 (OMIM 600993) was identified by exome sequencing as the disease gene mutated in MYHRS. Previously only three missense mutations affecting Ile500 (p.Ile500Thr, p.Ile500Val, and p.Ile500Met) have been described in 22 unrelated subjects with MYHRS or a clinically related phenotype. Here we report on a 15-year-old boy with typical MYHRS and a novel heterozygous SMAD4 missense mutation affecting residue Arg496. This finding provides further information about the distinctive SMAD4 mutation spectrum in MYHRS. In silico structural analyses exploring the impact of the Arg-to-Cys change at codon 496 suggested that conformational changes promoted by replacement of Arg496 impact the stability of the SMAD heterotrimer and/or proper SMAD4 ubiquitination.

Structural basis for the cooperative DNA recognition by Smad4 MH1 dimers.

Smad proteins form multimeric complexes consisting of the 'common partner' Smad4 and receptor regulated R-Smads on clustered DNA binding sites. Deciphering how pathway specific Smad complexes multimerize on DNA to regulate gene expression is critical for a better understanding of the cis-regulatory logic of TGF-ß and BMP signaling. To this end, we solved the crystal structure of the dimeric Smad4 MH1 domain bound to a palindromic Smad binding element. Surprisingly, the Smad4 MH1 forms a constitutive dimer on the SBE DNA without exhibiting any direct protein-protein interactions suggesting a DNA mediated indirect readout mechanism. However, the R-Smads Smad1, Smad2 and Smad3 homodimerize with substantially decreased efficiency despite pronounced structural similarities to Smad4. Therefore, intricate variations in the DNA structure induced by different Smads and/or variant energetic profiles likely contribute to their propensity to dimerize on DNA. Indeed, competitive binding assays revealed that the Smad4/R-Smad heterodimers predominate under equilibrium conditions while R-Smad homodimers are least favored. Together, we present the structural basis for DNA recognition by Smad4 and demonstrate that Smad4 constitutively homo- and heterodimerizes on DNA in contrast to its R-Smad partner proteins by a mechanism independent of direct protein contacts.

Negative regulation of TGFß signaling by the kinase LKB1 and the scaffolding protein LIP1.

Signal transduction by the Smad pathway elicits critical biological responses to many extracellular polypeptide factors, including TGFß and bone morphogenetic protein. Regulation of Smad signaling imparts several cytoplasmic and nuclear mechanisms, some of which entail protein phosphorylation. Previous work established a protein complex between Smad4 and the scaffolding protein LKB1-interacting protein 1 (LIP1). LKB1 is a well studied tumor suppressor kinase that regulates cell growth and polarity. Here, we analyzed the LKB1-LIP1 and the Smad4-LIP1 protein complexes and found that LIP1 can self-oligomerize. We further demonstrate that LKB1 is capable of phosphorylating Smad4 on Thr(77) of its DNA-binding domain. LKB1 inhibits Smad4 from binding to either TGFß- or bone morphogenetic protein-specific promoter sequences, which correlates with the negative regulatory effect LKB1 exerts on Smad4-dependent transcription. Accordingly, LKB1 negatively regulates TGFß gene responses and epithelial-mesenchymal transition. Thus, LKB1 and LIP1 provide negative control of TGFß signaling.

Hypomorph mutation-directed small-molecule protein-protein interaction inducers to restore mutant SMAD4-suppressed TGF-ß signaling.

Tumor suppressor genes represent a major class of oncogenic drivers. However, direct targeting of loss-of-function tumor suppressors remains challenging. To address this gap, we explored a variant-directed chemical biology approach to reverse the lost function of tumor suppressors using SMAD4 as an example. SMAD4, a central mediator of the TGF-ß pathway, is recurrently mutated in many tumors. Here, we report the development of a TR-FRET technology that recapitulated the dynamic differential interaction of SMAD4 and SMAD4R361H with SMAD3 and identified Ro-31-8220, a bisindolylmaleimide derivative, as a SMAD4R361H/SMAD3 interaction inducer. Ro-31-8220 reactivated the dormant SMAD4R361H-mediated transcriptional activity and restored TGF-ß-induced tumor suppression activity in SMAD4 mutant cancer cells. Thus, demonstration of Ro-31-8220 as a SMAD4R361H/SMAD3 interaction inducer illustrates a general strategy to reverse the lost function of tumor suppressors with hypomorph mutations and supports a systematic approach to develop small-molecule protein-protein interaction (PPI) molecular glues for biological insights and therapeutic discovery.

The functional differentiation of four smad4 paralogs in TGF-ß signaling pathway of Japanese flounder (Paralichthys olivaceus).

As a classical signaling pathway, transforming growth factor ß (TGF-ß) has been studied in various animals for more than decade years. However, the members of TGF-ß were markedly expanded in teleost specific third and fourth rounds of whole genome duplication (WGD). Here, four smad4s named Posmad4a, Posmad4b, Posmad4c and Posmad4d were identified in Japanese flounder. Our study showed that four flounder smad4s had distinct properties in terms of their protein structure, expression pattern, protein interaction and subcellular localization. PoSMAD4a/b were mainly located in the cytoplasm, and could co-localize in the nucleus with PoSMAD3a after TGF-ß activator stimulation. PoSMAD4c was mainly located in nucleus, whereas PoSMAD4d distributed in the whole cell. Both PoSMAD4c and PoSMAD4d could co-localize in the nucleus with PoSMAD3b after TGF-ß activator stimulation. Furthermore, Posmad4c responded most strongly to TGF-ß signal stimulation. Dual-luciferase reporter assay also showed that Posmad4c could specifically up-regulate the TGF-ß signal luciferase reporter gene, Posmad4b could enhance Wnt signal luciferase reporter gene, while both Posmad4b and Posmad4d could markedly up-regulate Notch signal reporter gene. All results indicated that Posmad4a/b/c/d had significantly functional differences among TGF-ß, Notch and Wnt signaling pathways. Our study provided important understanding to the biology of smad4s and its pathway crosstalk in teleost.

O-GlcNAc stabilizes SMAD4 by inhibiting GSK-3ß-mediated proteasomal degradation.

O-linked ß-N-acetylglucosamine (O-GlcNAc) is a post-translational modification which occurs on the hydroxyl group of serine or threonine residues of nucleocytoplasmic proteins. It has been reported that the presence of this single sugar motif regulates various biological events by altering the fate of target proteins, such as their function, localization, and degradation. This study identified SMAD4 as a novel O-GlcNAc-modified protein. SMAD4 is a component of the SMAD transcriptional complex, a major regulator of the signaling pathway for the transforming growth factor-ß (TGF-ß). TGF-ß is a powerful promoter of cancer EMT and metastasis. This study showed that the amount of SMAD4 proteins changes according to cellular O-GlcNAc levels in human lung cancer cells. This observation was made based on the prolonged half-life of SMAD4 proteins. The mechanism behind this interaction was that O-GlcNAc impeded interactions between SMAD4 and GSK-3ß which promote proteasomal degradation of SMAD4. In addition, O-GlcNAc modification on SMAD4 Thr63 was responsible for stabilization. As a result, defects in O-GlcNAcylation on SMAD4 Thr63 attenuated the reporter activity of luciferase, the TGF-ß-responsive SMAD binding element (SBE). This study's findings imply that cellular O-GlcNAc may regulate the TGF-ß/SMAD signaling pathway by stabilizing SMAD4.

MH1 domain of SMAD4 binds N-terminal residues of the homeodomain of Hoxc9.

Smad family proteins mediate signaling initiated by bone morphogenetic proteins (BMPs). Upon BMP stimulation, the Smads such as Smad4 can interact directly with Hox proteins and suppress their DNA-binding activity. Although the interaction between the MAD-homology 1 (MH1) domain of Smad4 and Hox was found to regulate the transcription activity of Hox proteins, the molecular mechanism is not well characterized and direct contact residues remain to be elucidated. In the present study, the interaction between the recombinant homeodomain (HD) of Hoxc9 and MH1 domain of Smad4 was investigated with the use of the GST pull-down assay, surface plasmon resonance (SPR) analysis as well as multidimensional nuclear magnetic resonance (NMR) techniques. The Hoxc9-HD was precipitated with the GST-fused Smad4-MH1 but not with GST alone, demonstrating a direct interaction between Hoxc9-HD and Smad4-MH1 in vitro. SPR measurement further confirmed a moderately strong interaction (K(d) approximately 400 nM) between these two domains. Moreover, NMR titration experiments showed that a strong and specific binding occurred between Smad4-MH1 and Hoxc9-HD. NMR triple-resonance experiments and backbone assignments revealed that the N-terminal arm of Hoxc9-HD, spanning the positive-charged DNA-binding segment of Arg190-Arg196, was intimately involved in the interaction with Smad4-MH1. Ala-substitutions of Arg190-Arg196 led to the loss of interaction between Hoxc9-HD and Smad4-MH1 in both GST-pull down assay and SPR analysis; further provided functional evidence for the critical role of this positive-charged region in binding to Smad4-MH1. This suggested that Smad4-MH1 could occupy one of the DNA binding sites of Hoxc9 and consequently inhibits its transcription activity. The above results are in good agreement and yield the first insight into the interaction between the homeodomain of Hox proteins and the conserved MH1 domain of Smad family proteins.

Biophysical simulations and structure-based modeling of residue interaction networks in the tumor suppressor proteins reveal functional role of cancer mutation hotspots in molecular communication.

In the current study, we have combined molecular simulations and energetic analysis with dynamics-based network modeling and perturbation response scanning to determine molecular signatures of mutational hotspot residues in the p53, PTEN, and SMAD4 tumor suppressor proteins. By examining structure, energetics and dynamics of these proteins, we have shown that inactivating mutations preferentially target a group of structurally stable residues that play a fundamental role in global propagation of dynamic fluctuations and mediating allosteric interaction networks. Through integration of long-range perturbation dynamics and network-based approaches, we have quantified allosteric potential of residues in the studied proteins. The results have revealed that mutational hotspot sites often correspond to high centrality mediating centers of the residue interaction networks that are responsible for coordination of global dynamic changes and allosteric signaling. Our findings have also suggested that structurally stable mutational hotpots can act as major effectors of allosteric interactions and mutations in these positions are typically associated with severe phenotype. Modeling of shortest inter-residue pathways has shown that mutational hotspot sites can also serve as key mediating bridges of allosteric communication in the p53 and PTEN protein structures. Multiple regression models have indicated that functional significance of mutational hotspots can be strongly associated with the network signatures serving as robust predictors of critical regulatory positions responsible for loss-of-function phenotype. The results of this computational investigation are compared with the experimental studies and reveal molecular signatures of mutational hotspots, providing a plausible rationale for explaining and localizing disease-causing mutations in tumor suppressor genes.

Expression and potential regulation of miRNA­431 during lung development of Sprague­Dawley rats.

Deficiency of surfactant proteins (SPs) is the main cause of respiratory distress syndrome (RDS) and chronic lung diseases. Our previous study demonstrated that miR­431 was differentially expressed between infants with RDS and infants without RDS using microarray analysis. However, the potential role of miR­431 in the development of lung function is still unknown. In the present study, the morphological characteristics of lung tissues and the expression levels of miR­431 were examined at three time points of rat lung development [gestational days 19 and 21 (E19, and E21) and postnatal day (P3)]. The protein and mRNA levels of SMAD4 and SPs (SP­A, SP­B, SP­C and SP­D) were also validated by reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and western blot analysis, respectively. The expression levels of miR­431 were gradually decreased over time periods of E19, E21 and P3, as determine using RT­qPCR and fluorescence in situ hybridization. Dual luciferase­reporter assays revealed that SMAD4 is a direct target of miR­431. The mRNA and protein expression levels of SMAD4 and SPs increased gradually in rat lung tissues from E19 to P3. The order of magnitude was as follows: E19, E21 and P3. The present study demonstrated that the expression level of miR­431 decreased in the order of E19, E21 and P3 during rat lung development. The target gene of miR­431, SMAD4, was negatively regulated by miR­431, and its expression levels in the rat lung tissue increased from E19 to the P3. Surfactant synthesis was further increased over the E19 to P3 time period. Further studies are required to determine how miR­431 regulates pulmonary surfactant synthesis by targeting SMAD4.

Computational investigation of a ternary model of SnoN-SMAD3-SMAD4 complex.

The transforming growth factor ß (TGFß) plays an essential role in the regulation of cellular processes such as cell proliferation, migration, differentiation, and apoptosis by association with SMAD transcriptional factors that are regulated by the transcriptional regulator SnoN. The crystal structure of SnoN-SMAD4 reveals that SnoN can adopt two binding modes, the open and closed forms, at the interfaces of SMAD4 subunits. Accumulating evidence indicates that SnoN can interact with both SMAD3 and SMAD4 to form a ternary SnoN-SMAD3-SMAD4 complex in the TGFß signaling pathway. However, how the interaction of SnoN with the SMAD3 and SMAD4 remains unclear. Here, molecular dynamics (MD) simulations and molecular modeling methods were performed to figure out this issue. The simulations reveal that SnoNopen exists in two, open and semi-closed, conformations. Molecular modeling and MD simulation studies suggest that the SnoNclosed form interferes with the SMAD3-SMAD4 protein; in contract, the SnoNopen can form a stable SnoN-SMAD3-SMAD4 complex. These mechanistic mechanisms may help elucidate the detailed engagement of SnoN with two SMAD3 and SMAD4 transcriptional factors in the regulation of TGFß signaling pathway.

Potentiation of Smad-mediated transcriptional activation by the RNA-binding protein RBPMS.

Smad2, Smad3 and Smad4 proteins are considered to be key mediators of transforming growth factor-beta (TGF-beta) signaling. However, the identities of the Smad partners mediating TGF-beta signaling are not fully understood. Here, we show that RNA-binding protein with multiple splicing (RBPMS), a member of the RNA-binding protein family, physically interacts with Smad2, Smad3 and Smad4 both in vitro and in vivo. The presence of TGF-beta increases the binding of RBPMS with these Smad proteins. Consistent with the binding results, overexpression of RBPMS enhances Smad-dependent transcriptional activity in a TGF-beta-dependent manner, whereas knockdown of RBPMS decreases this activity. RBPMS interacts with TGF-beta receptor type I (TbetaR-I), increases phosphorylation of C-terminal SSXS regions in Smad2 and Smad3, and promotes the nuclear accumulation of the Smad proteins. Moreover, RBPMS fails to enhance the transcriptional activity of Smad2 and Smad3 that lack the C-terminal phosphorylation sites. Our data provide the first evidence for an RNA-binding protein playing a role in regulation of Smad-mediated transcriptional activity and suggest that RBPMS stimulates Smad-mediated transactivation possibly through enhanced phosphorylation of Smad2 and Smad3 at the C-terminus and promotion of the nuclear accumulation of the Smad proteins.

MicroRNA-574 enhances doxorubicin resistance through down-regulating SMAD4 in breast cancer cells.

OBJECTIVE: Drug resistance has become an important factor that threatens the survival and prognosis of patients with breast cancer, especially in patients with advanced breast cancer. Several microRNAs have been proved to participate in the resistant process; however, the role of miR-574 in doxorubicin (Dox) resistant breast cancer is still unclear. PATIENTS AND METHODS: Quantitative Real-time poly chain reaction (qRT-PCR) was employed to detect the expression level of miR-574 in breast cancer Dox-resistant MCF-7/Adr cell line and parental MCF-7 cell line. Using miR-574 mimics and inhibitors, miR-574 level was up- or down- regulated. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was handled to detect the IC50, and flow cytometric analysis was employed to measure the apoptosis and cell circle. Dual-luciferase and Western-blot experiments were applied to verify the direct target gene of miR-574. RESULTS: miR-574 expression level was significantly higher in MCF-7/Adr cells compared to normal MCF-7 cells. Up-regulation of miR-574 level in MCF-7 cells promoted the cell growth and G0/G1-to-S phase transition but inhibited cell apoptosis. However, knockdown of miR-574 in MCF-7/Adr cells decreased the IC50 and cell growth. Using luciferase assay, SMAD4 was confirmed to be a potential target of miR-574, and the expression of SMAD4 protein was regulated by miR-574. In blood samples of patients, the miR-574 level before chemotherapy was higher than that after chemotherapy. CONCLUSIONS: We revealed miR-574 could promote doxorubicin resistance of breast cancer MCF-7 cells via down-regulating SMAD4, thus providing a novel target for advancing breast cancer chemotherapy.