Growth-regulated expression and G0-specific turnover of the mRNA that encodes URH49, a mammalian DExH/D box protein that is highly related to the mRNA export protein UAP56

URH49 is a mammalian protein that is 90% identical to the DExH/D box protein UAP56, an RNA helicase that is important for splicing and nuclear export of mRNA. Although Saccharomyces cerevisiae and Drosophila express only a single protein corresponding to UAP56, mRNAs encoding URH49 and UAP56 are both expressed in human and mouse cells. Both proteins interact with the mRNA export factor Aly and both are able to rescue the loss of Sub2p (the yeast homolog of UAP56), indicating that both proteins have similar functions. UAP56 mRNA is more abundant than URH49 mRNA in many tissues, although in testes URH49 mRNA is much more abundant. UAP56 and URH49 mRNAs are present at similar levels in proliferating cultured cells. However, when the cells enter quiescence, the URH49 mRNA level decreases 3–6-fold while the UAP56 mRNA level remains relatively constant. The amount of URH49 mRNA increases to the level found in proliferating cells within 5 h when quiescent cells are growth-stimulated or when protein synthesis is inhibited. URH49 mRNA is relatively unstable (T_½ = 4 h) in quiescent cells, but is stabilized immediately following growth stimulation or inhibition of protein synthesis. In contrast, there is much less change in the content or stability of UAP56 mRNA following growth stimulation. Our observations suggest that in mammalian cells, two UAP56-like RNA helicases are involved in splicing and nuclear export of mRNA. Differential expression of these helicases may lead to quantitative or qualitative changes in mRNA expression.     

Induced expression of miR-1250-5p exerts tumor suppressive role in triple-negative breast cancer cells

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, and it has a prevalence rate of 15%–20% among all breast cancer cases in younger women. Still, the underlying molecular mechanisms of its pathogenesis are not entirely understood. In the previous study, we identified that microRNA (miR)-1250-5p is significantly down-expressed in TNBC cells. Thus, in the present study, we explore the functional anticancer role of miR‑1250‑5p in the transient mimic transfected TNBC cells. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to examine the effect of miR-1250-5p on cell viability of TNBC (MDA-MB-231 and MDA-MB-453) cells. The confocal microscopy, quantitative real-time polymerase chain reaction, and western blot analysis techniques were used to assess the effect of miR-1250-5p on cancer hallmarks in test cells. Induced miR‑1250-5p expression in MDA-MB-231 and MDA-MB-453 cells decreased cell viability in a time-dependent manner. Increased miR‑1250-5p expression levels significantly decreased cell cycle G1/S phase transition markers (Cyclin D1 and CDK4) at messenger RNA (mRNA) and protein levels in TNBC cells compared to scrambled sequence transfected cells. Transient transfection of TNBC cells with miR-1250-5p mimic increased apoptosis in TNBC cells by increasing the level of active caspase (Caspase 8 and Caspase 3) of the intrinsic pathway. Apoptosis-related morphological changes were also observed in the test cells. Further, the induced expression of miR-1250-5p significantly decreased epithelial-mesenchymal transition (EMT) by altering the mRNA and protein levels of E-cadherin and Vimentin. Moreover, results of confocal microscopy revealed increased reactive oxygen species generation, and decreased mitochondria membrane potential in miR-1250-5p mimic transient transfected TNBC cells. In conclusion, miR‑1250-5p acts as tumor suppressor in TNBC cells and its induction by therapeutics might be a novel strategy for the disease treatment.     

Inhibition of SK4 Potassium Channels Suppresses Cell Proliferation,Migration and the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells

Treatments for triple-negative breast cancer (TNBC) are limited; intermediate-conductance calcium-activated potassium (SK4) channels are closely involved in tumor progression, but little is known about these channels in TNBC. We aimed to investigate whether SK4 channels affect TNBC. First, by immunohistochemistry (IHC) and western blotting (WB), increased SK4 protein expression in breast tumor tissues was detected relative to that in non-tumor breast tissues, but there was no apparent expression difference between various subtypes of breast cancer (p>0.05). Next, functional SK4 channels were detected in the TNBC cell line MDA-MB-231 using WB, real-time PCR, immunofluorescence and patch-clamp recording. By employing SK4 specific siRNAs and blockers, including TRAM-34 and clotrimazole, in combination with an MTT assay, a colony-formation assay, flow cytometry and a cell motility assay, we found that the suppression of SK4 channels significantly inhibited cell proliferation and migration and promoted apoptosis in MDA-MB-231 cells (p<0.05). Further investigation revealed that treatment with epidermal growth factor (EGF)/basic fibroblast growth factor (bFGF) caused MDA-MB-231 cells to undergo the epithelial-mesenchymal transition (EMT) and to show increased SK4 mRNA expression. In addition, the down-regulation of SK4 expression inhibited the EMT markers Vimentin and Snail1. Collectively, our findings suggest that SK4 channels are expressed in TNBC and are involved in the proliferation, apoptosis, migration and EMT processes of TNBC cells.     

Sulforaphane-cisplatin combination inhibits the stemness and metastatic potential of TNBCs via down regulation of sirtuins-mediated EMT signaling axis

BackgroundSulforaphane (SFN) is a naturally occurring organosulfur compound found in cruciferous vegetables such as broccoli, brussels sprouts and cabbage. SFN is known for its multiple therapeutic properties, such as HDAC inhibitory, chemo preventive and anti-cancer effects. Cisplatin (CIS) has limited effect against metastatic triple-negative breast cancer (TNBC). Additionally, CIS impose severe side effects to normal cells, and later TNBC cells develops resistance. Studies suggest that the overexpression of sirtuins (SIRTs) promotes CIS resistance and metastasis by activating epithelial-to-mesenchymal transition (EMT) pathway in TNBC.PurposeIn view of the above information, we investigated the therapeutic efficacy of SFN, in combination with CIS against TNBC metastasis and CIS resistance.MethodsThe anti-cancerous effect of SFN-CIS combination on human TNBC cell lines was demonstrated by utilizing MTT assay and, apoptosis and cell cycle assay followed by FACS analysis. The synergistic effect of SFN-CIS combination on the experimental metastasis was demonstrated by utilizing migration, invasion, chemotaxis, mammosphere and colony formation assay on human TNBC MDA-MB-231 and MDA-MB-468 cells. The role of SIRTs-mediated EMT signaling axis in the metastasis and chemoresistance was investigated by western blotting technique as well as sirtuin activity tests. This was further validated by using Chromatin immunoprecipitation (ChIP) analysis.ResultsWe found that SFN-CIS combination synergistically inhibits cellular growth of MDA-MB-231 and MDA-MB-468 cells. More importantly, SFN was found to protect normal kidney cells from CIS-induced toxicity. Further, SFN-CIS combination was found to synergistically inhibit metastatic-events via significantly altering EMT markers which was further associated with the suppression of SIRTs functions in TNBC cells. ChIP analysis validated that SFN-CIS combination suppresses EMT mechanism through altered chromatin modifications at E-cadherin promoter resulting in its re-expression.ConclusionThe results of the current study suggests that CIS when supplemented with SFN, inhibits metastasis and stemness potential of TNBC cells by down regulating SIRTs-mediated EMT cascade. Overall this study affirms that, this novel combination could be a promising strategy against SIRT-mediated TNBC metastasis and CIS-resistance.     

Biochemical characterization of the ATPase and helicase activity of UAP56, an essential pre-mRNA splicing and mRNA export factor

DEXD/H-box protein UAP56 is an essential pre-mRNA splicing factor required for the first ATP-dependent spliceosome assembly step. UAP56 is also essential for the export of the majority of mRNAs from the nucleus to the cytoplasm. We performed biochemical characterization of UAP56's ATPase and helicase activity, which is important for further understanding the role of these activities in UAP56's function. We showed that UAP56 is an RNA-stimulated ATPase that can only hydrolyze ATP. We demonstrated that UAP56 is an ATP-dependent RNA helicase that can unwind substrates with 5' or 3' overhangs or blunt ends in vitro. We showed that U2AF(65) and Aly, two proteins known to interact with UAP56, do not influence UAP56's ATPase or helicase activity. We also demonstrated that several mutants in the conserved helicase motifs I, II, and III abolish UAP56's ATPase and/or helicase activity, providing tools for future investigation of the role of UAP56's ATPase and helicase activity in spliceosome assembly and mRNA export.     

UAP56 levels affect viability and mRNA export in Caenorhabditis elegans

Expression of a gfp transgene in the intestines of living Caenorhabditis elegans has been measured following depletion by RNAi of a variety of known splicing factors and mRNA export proteins. Reduction of most splicing factors showed only a small effect on expression of the transgene in the animal injected with dsRNA, although most of these RNAi's resulted in embryonic lethality in their offspring. In contrast, RNAi of nxf-1, the worm homolog of mammalian NXF1/TAP, a key component of the mRNA export machinery, resulted in dramatic suppression of GFP expression in the injected animals. When we tested other proteins previously reported to be involved in marking mRNAs for export, we obtained widely divergent results. Whereas RNAi of the worm REF/Aly homologs had no obvious effect, either in the injected animals or their offspring, RNAi of UAP56, reported to be the partner of REF/Aly, resulted in strong suppression of GFP expression due to nuclear retention of its mRNA. Overexpression of UAP56 also resulted in rapid loss of GFP expression and lethality at all stages of development. We conclude that UAP56 plays a key role in mRNA export in C. elegans, but that REF/Aly may not. It also appears that some RNA processing factors are required for viability (e.g., U2AF, PUF60, SRp54, SAP49, PRP8, U1-70K), whereas others are not (e.g., U2A', CstF50).     

Eribulin upregulates miR-195 expression and downregulates Wnt3a expression in non-basal-like type of triple-negative breast cancer cell MDA-MB-231

Triple-negative breast cancer (TNBC), which does not show hormone sensitivity, is a poor prognosis disease without an established targeted treatment, so that establishing a therapeutic target for each subtype is desired. In addition, microRNA (miRNA), a non-cording RNA 19–25 nucleotide-longs in length, is known to be involved in regulating gene expression. We examined miRNA expression after exposure to eribulin, MDA-MB-231 cells, non-basal-like type of TNBC cell lines, and HCC1143 cells, basal-like type of TNBC cell lines. The activity of caspase-3 significantly increased compared to the control in MDA-MB-231, whereas no significant difference was observed in HCC1143. The expression level of 20-miRNAs significantly increased compared to the control in MDA-MB-231 after exposure to eribulin. The expression level of 6-miRNAs also significantly increased compared to the control in HCC1143. In these 2 cell types, miR-125b-1 and miR-195 were commonly expressed. While the expression level of miR-125b-1 decreased in both cells, the expression level of miR-195 increased in MDA-MB-231 and decreased in HCC1143. The expression level of miR-195 targeting Wnt3a significantly decreased compared to the control in MDA-MB-231, whereas it significantly increased in HCC1143. These results showed that exposure to eribulin highly increased the expression of miR-195 while it decreased the expression of Wnt3a in non-basal-like type of TNBC. Some miRNAs are known to regulate other signaling pathways involved in human pathogenesis by regulating the Wnt signaling pathway, and miRNA can act as a tumor-suppressing gene; therefore, miR-195 may serve as a therapeutic target in non-basal-like type of TNBC.     

The monoamine oxidase-A inhibitor clorgyline promotes a mesenchymal-to-epithelial transition in the MDA-MB-231 breast cancer cell line

Monoamine oxidase-A (MAO-A) dysfunction has been historically associated with depression. Recently, depression as well as altered MAO-A expression have both been associated with a poor prognosis in cancers, although the mechanism involved remains ambiguous. For example, MAO-A mRNA is repressed across cancers, yet MAO-A protein and levels of serotonin, a substrate of MAO-A implicated in depression, are paradoxically increased in malignancies, including breast cancer.The effect of clorgyline (CLG), a selective inhibitor of MAO-A, on malignant behaviour, expression of transitional markers, and biochemical correlates was examined in two human breast carcinoma cell lines, i.e. the epithelial, oestrogen receptor (ER)-positive MCF-7 cell line and the post-EMT (mesenchymal), ER-negative MDA-MB-231 cell line.CLG exerted little effect on malignant behaviour in MCF-7 cells, but inhibited proliferation and anchorage-independent growth, and increased invasiveness and active migration of MDA-MB-231 cells. CLG induced the expression of the mesenchymal marker vimentin in MCF-7 cells, but not in MDA-MB-231 cells. In contrast, CLG induced the epithelial protein marker E-cadherin in both cell lines, with a more robust effect in MDA-MB-231 cells (where a nuclear E-cadherin signal was also detected). This effect appears to be independent of any canonical Snai1-mediated regulation of E-cadherin mRNA expression. CLG interfered with the β-catenin/[phospho]GSK-3β complex as well as the E-cadherin/β-catenin complex in both cell lines cells, but, again, the effect was more robust in MDA-MB-231 cells. Parallel studies revealed a general lack of effect of CLG on the ER-negative, epithelial Au565 breast cancer cell line. Thus, any effect of CLG on metastatic behaviours appears to rely on the cell's EMT status rather than on the cell's ER status.These data suggest that inactivation of MAO-A triggers a mesenchymal-to-epithelial transition in MDA-MB-231 cells via a non-canonical mechanism. This potentially implicates an MAO-A-sensitive step in advanced breast cancer and should be borne in mind when considering pharmacological treatment options for co-morbid depression in breast cancer patients.     

Metallothionein-3 Increases Triple-Negative Breast Cancer Cell Invasiveness via Induction of Metalloproteinase Expression

It has been recently found that metallothionein-3 (MT3) enhances the invasiveness and tumorigenesis of prostate cancer cells. This finding is in contrast to those of earlier studies, which indicated that overexpression of MT3 in breast cancer and prostate cancer cell lines inhibits their growth in vitro. Therefore, to clarify the role of MT3 in breast cancer progression, we analyzed the effect of MT3-overexpression on proliferation, invasiveness, migration, and tumorigenesis of breast cancer MDA-MB-231/BO2 cells. It was found that MDA-MB-231/BO2 cells overexpressing MT3 were characterized by increased invasiveness in vitro, compared to the control cells. Interestingly, this increased invasiveness correlated with a highly increased concentration of MMP3 in the culture supernatants (p<0.0001). Our data suggest that MT3 may regulate breast cancer cell invasiveness by modulating the expression of MMP3. These experimental results, obtained using triple-negative MDA-MB-231/BO2 cells, were further supported by clinical data. It was found that, in triple-negative breast cancer (TNBC), nuclear MT3 immunoreactivity in cancer cells tended to be associated with patients’ shorter disease-specific survival, suggesting that nuclear MT3 expression may be a potential marker of poor prognosis of triple-negative TNBC cases.     

A loop involving NRF2, miR-29b-1-5p and AKT,regulates cell fate of MDA-MB-231 triple-negative breast cancer cells

The present study shows that nuclear factor erythroid 2-related factor 2 (NRF2) and miR-29b-1-5p are two opposite forces which could regulate the fate of MDA-MB-231 cells, the most studied triple-negative breast cancer (TNBC) cell line. We show that NRF2 activation stimulates cell growth and markedly reduces reactive oxygen species (ROS) generation, whereas miR-29b-1-5p overexpression increases ROS generation and reduces cell proliferation. Moreover, NRF2 downregulates miR-29b-1-5p expression, whereas miR-29b-1-5p overexpression decreases p-AKT and p-NRF2. Furthermore, miR-29b-1-5p overexpression induces both inhibition of DNA N-methyltransferases (DNMT1, DNMT3A, and DNMT3B) expression and re-expression of HIN1, RASSF1A and CCND2. Conversely, NRF2 activation induces opposite effects. We also show that parthenolide, a naturally occurring small molecule, induces the expression of miR-29b-1-5p which could suppress NRF2 activation via AKT inhibition. Overall, this study uncovers a novel NRF2/miR-29b-1-5p/AKT regulatory loop that can regulate the fate (life/death) of MDA-MB-231 cells and suggests this loop as therapeutic target for TNBC.     

UAP56 is an important regulator of protein synthesis and growth in cardiomyocytes

UAP56, an ATP dependent RNA helicase that also has ATPase activity, is a DExD/H box protein that is phylogenetically grouped with the eukaryotic initiation factor eIF4A, the prototypical member of the DExD/H box family of helicases. UAP56, also known as BAT1, is an essential RNA splicing factor required for spliceosome assembly and mRNA export but its role in protein synthesis is not known. Here we demonstrate that UAP56 regulates protein synthesis and growth in cardiomyocytes. We found that wild-type (WT) UAP56 increased serum induced protein synthesis in HeLa cells. UAP56 mutants lacking ATPase and/or helicase activity inhibited protein synthesis compared with WT UAP56, suggesting that the ATPase and RNA helicase activity of UAP56 is important for protein synthesis. UAP56 siRNA inhibited phenylephrine (PE) induced protein synthesis in cardiomyocytes and inhibited PE induced cardiomyocyte hypertrophy. Our data demonstrate that UAP56 is an important regulator of protein synthesis and plays an important role in the regulation of cardiomyocyte growth.     

ANXA2 could act as a moderator of EGFR-directed therapy resistance in triple negative breast cancer

Triple negative breast cancer (TNBC) patients cannot benefit from EGFR-targeted therapy even though the EGFR is highly expressed, because patients exhibit resistance to these drugs. Unfortunately, the molecular mechanisms remain relatively unknown. ANXA2, highly expressed in invasive breast cancer cells, is closely related with poor prognosis, and acts as a molecular switch to EGFR activation. In this study, MDA-MB-231 cells and MCF7 cells were used. Our results showed that ANXA2 expression is inversely correlated with cell sensitivity to gefitinib. Knockdown of ANXA2 expression in MDA-MB-231 cells increased the gefitinib induced cell death. When ANXA2 was overexpressed in MCF7 cells, the gefitinib induced cell death was decreased. Furthermore, we demonstrated that phosphorylation of ANXA2 at Tyr23 is negatively correlated with the sensitivity of TNBC to gefitinib. Altogether, our results suggest a new role of ANXA2 in regulating sensitivity of TNBC MDA-MB-231 cells to the EGFR inhibitor gefitinib.     

ROR2 promotes the epithelial-mesenchymal transition by regulating MAPK/p38 signaling pathway in breast cancer

Receptor tyrosine kinase-like orphan receptor 2 (ROR2) is a tyrosine-protein kinase receptor highly implicated in the growth plate and cartilage development, which may be involved in epithelial-mesenchymal transition (EMT) in breast cancer (BC) cells. Although ROR2 is known to promote the migration of BC cells, the detailed mechanism of this event is still not clear. Here, we found that ROR2 expression was significantly increased in BC lymphatic metastatic tissue as well as BC samples compared to normal adjacent breast tissues. A higher expression of ROR2 in MDA-MB-231 and a lower expression of ROR2 in MCF-7 cells were observed. MDA-MB-231-siROR2 cells with ROR2 knockdown inhibited MDA-MB-231 cell invasion, migration, and clonal formation, while MCF-7-OvROR2 cells with overexpression showed the opposite results. The underlying mechanisms involved in ROR2-induced EMT in MDA-MB-231 and MCF-7 cells were further investigated. ROR2 may activate EMT progression in BC cells by altering MAPK kinase 3/6 (MKK3/6) expression. The expressions of transforming growth factor-β, matrix metalloproteinase-2 (MMP-2), and MMP-9, which were related to tumor cell invasion activities, were notably increased in MCF-7-OvROR2 cells. The EMT markers, including snail, N-cadherin, tissue inhibitor of metalloproteinases-1, and vimentin, were significantly upregulated in MCF-7-OvROR2 cells. On the contrary, E-cadherin was obviously reduced expressed in MCF-7-OvROR2 cells. ROR2 may regulate the malignant phenotype of BC cells possibly via activation of mitogen-activated protein kinase (MAPK)/p38 signaling pathway. Collectively, ROR2 promotes BC carcinogenesis by mediating the MAPK/p38 pathway, which is independent of Wnt5α.