Chromosome Region Maintenance Research Articles

Conserved methionine 165 of matrix protein contributes to the nuclear import and is essential for influenza A virus replication

BackgroundThe influenza matrix protein (M1) layer under the viral membrane plays multiple roles in virus assembly and infection. N-domain and C-domain are connected by a loop region, which consists of conserved RQMV motif.MethodsThe function of the highly conserve RQMV motif in the influenza virus life cycle was investigated by site-directed mutagenesis and by rescuing mutant viruses by reverse genetics. Co-localization of M1 with nucleoprotein (NP), clustered mitochondria homolog protein (CLUH), chromosome region maintenance 1 protein (CRM1), or plasma membrane were studied by confocal microscopy.ResultsMutant viruses containing an alanine substitution of R163, Q164 and V166 result in the production of the virus indistinguishable from the wild type phenotype. Single M165A substitution was lethal for rescuing infection virus and had a striking effect on the distribution of M1 and NP proteins. We have observed statistically significant reduction in distribution of both M165A (p‹0,05) and NP (p‹0,001) proteins to the nucleus in the cells transfected with the reverse –genetic system with mutated M1. M165A protein was co-localized with CLUH protein in the cytoplasm and around the nucleus but transport of M165-CLUH complex through the nuclear membrane was restricted.ConclusionsOur finding suggest that methionine 165 is essential for virus replication and RQMV motif is involved in the nuclear import of viral proteins.

Nuclear transport inhibition in acute myeloid leukemia: recent advances and future perspectives.

Selective inhibitors of nuclear export (SINE) are emerging as a potentially efficacious therapeutic strategy for overcoming resistance to conventional chemotherapy for acute myeloid leukemia. SINE specifically block the protein Exportin 1, also known as chromosomal region maintenance 1, leading to nuclear retention of cargo proteins, including several tumor suppressor proteins. Selinexor, a first generation SINE, is currently in early phase clinical studies in various combinations with promising antileukemic and pro-apoptotic activity. Here we discuss the mechanism of action of SINEs and further elaborate on the clinical data available from the various trials in acute myeloid leukemia.

Differential expression and molecular interactions of chromosome region maintenance 1 and calreticulin exportins in breast cancer cells

Chromosome region maintenance 1 (CRM-1) and calreticulin (CALR) are two proteins that act as exportins for some nuclear receptors, in addition to other critical functions for cellular homeostasis. In several cancer types, CRM-1 and CALR are upregulated suggesting an imbalance in their functions. However, the regulation of CRM-1 and CALR, and their biological implications, are not completely known. Here, we evaluated the interplay between the levels of CRM-1 and CALR, and estrogen receptor alpha (ERα) status, in breast cancer cells. CRM-1 and CALR were upregulated in mammary tumors relative to normal mammary tissue. Furthermore, the mRNA and protein levels of CRM-1 and CALR were higher in breast cancer cells lacking ERα, in comparison with those that express ERα. Additionally, both proteins were distributed in the nucleus and cytoplasm in the two cell types. Importantly, we identified novel interactions for these exportins. First, we showed an interaction between CRM-1 and CALR, and then we identified that SUN1 and SUN2, two proteins localized in the nuclear envelop, were able to interact specifically with CRM-1, but not CALR. Interestingly, SUN1 and SUN2 expression seemed to be decreased in breast cancer, thereby affecting the interactions of these proteins with CRM-1, and possibly its actions as an exportin. Thus, our data suggest that expression levels for CRM-1 and CALR, the interaction between these exportins, and specific interactions of SUN1 and SUN2 with CRM-1 but not CALR, may be central elements in nucleo-cytoplasmic transport. Furthermore, deregulation of these elements may have serious implications in the progression of breast and other types of cancer.

Phosphorylation and dephosphorylation of threonine 188 in nucleoprotein is crucial for the replication of influenza A virus

Nucleoprotein (NP) is a major component of the viral ribonucleoprotein (vRNP) complex that is responsible for viral replication, transcription and packaging of influenza A virus. Phosphorylation of NP plays an important role during viral infection. In the present study, we identified threonine 188 (T188) as a novel phosphorylated residue in the NP of influenza A virus by using mass spectrometry. T188 is located within nuclear export signal 2 (NES2) which is chromosome region maintenance 1 (CRM1)-independent. We observed that the phosphorylation and dephosphorylation of residue T188 regulated viral replication by controlling NES2-dependent NP nuclear export and the polymerase activity of the vRNP complex. Our findings provide further insights for understanding the replication of influenza A virus.

Nuclear Export Inhibition for Pancreatic Cancer Therapy.

Pancreatic cancer is a deadly disease that is resistant to most available therapeutics. Pancreatic cancer to date has no effective drugs that could enhance the survival of patients once their disease has metastasized. There is a need for the identification of novel actionable drug targets in this unusually recalcitrant cancer. Nuclear protein transport is an important mechanism that regulates the function of several tumor suppressor proteins (TSPs) in a compartmentalization-dependent manner. High expression of the nuclear exporter chromosome maintenance region 1 (CRM1) or exportin 1 (XPO1), a common feature of several cancers including pancreatic cancer, results in excessive export of critical TSPs to the incorrect cellular compartment, leading to their functional inactivation. Small molecule inhibitors of XPO1 can block this export, retaining very important and functional TSPs in the nucleus and leading to the effective killing of the cancer cells. This review highlights the current knowledge on the role of XPO1 in pancreatic cancer and how this serves as a unique and clinically viable target in this devastating and by far incurable cancer.

Intersectin goes nuclear: secret life of an endocytic protein

Intersectin 1-short (ITSN1-s) is a 1220 amino acid ubiquitously expressed scaffold protein presenting a multidomain structure that allows to spatiotemporally regulate the functional interaction of a plethora of proteins. Besides its well-established role in endocytosis, ITSN1-s is involved in the regulation of cell signaling and is implicated in tumorigenesis processes, although the signaling pathways involved are still poorly understood. Here, we identify ITSN1-s as a nucleocytoplasmic trafficking protein. We show that, by binding to importin (IMP)α, a small fraction of ITSN1-s localizes in the cell nucleus at the steady state, where it preferentially associates with the nuclear envelope and interacts with lamin A/C. However, upon pharmacological ablation of chromosome region maintenance 1 (CRM-1)-dependent nuclear export pathway, the protein accumulates into the nucleus, thus revealing its moonlighting nature. Analysis of deletion mutants revealed that the coiled coil (CC) and Src homology (SH3) regions play the major role in its nucleocytoplasmic shuttling. While no evidence of nuclear localization signal (NLS) was detected in the CC region, a functional bipartite NLS was identified within the SH3D region of ITSN1-s (RKKNPGGWWEGELQARGKKRQIGW-1127), capable of conferring energy-dependent nuclear accumulation to reporter proteins and whose mutational ablation affects nuclear import of the whole SH3 region. Thus, ITSN1-s is an endocytic protein, which shuttles between the nucleus and the cytoplasm in a CRM-1- and IMPα-dependent fashion.

Crm1 knockdown by specific small interfering RNA reduces cell proliferation and induces apoptosis in head and neck cancer cell lines.

Head and neck squamous cell carcinoma (HNSCC) is the most common and most aggressive type of head and neck cancer. Current approaches for the treatment of HNSCC are not sufficient to increase the patient survival or to reduce the high recurrence rate. Consequently, there is a need to explore the molecular characteristics of this cancer in order to discover potential therapeutic target molecules. The overexpression of chromosome region maintenance 1 (Crm1), responsible for the transport of different classes of macromolecules from the nuclear membrane to the cytoplasm, in various cancer cells has made it an attractive target molecule in cancer research. It has been reported that transcription factors, which are the target cargo proteins of Crm1, have critical roles in regulating intracellular processes via their expression levels and functions, which in turn are regulated by the cell cycle and signaling proteins. Previous findings show that head and neck cancer cells overexpress Crm1 and that these cells become highly dependent on Crm1 function. The results of this study show that after decreasing Crm1 expression levels in HNSCC cells through either treatment with specific Crm1 RNA interference (siRNA) or the selective Crm1 inhibitor leptomycin B (LMB), cell viability, proliferation, migration, and wound-healing abilities decreased, suppressing tumorigenic properties through the induction of apoptosis. Crm1 is a powerful diagnostic biomarker because of its central role in cancerogenesis, and it has a high potential for the development of targeted Crm1 molecules or synthetic agents, such as LMB, as well as for the improvement of the clinical features in head and neck cancer.

KPT-330 inhibition of chromosome region maintenance 1 is cytotoxic and sensitizes chronic myeloid leukemia to Imatinib

As tyrosine kinase inhibitors (e.g., Imatinib, IM) fail to induce long-term response in some chronic myeloid leukemia (CML), novel therapies targeting leukemia-dysregulated pathways are necessary. Nuclear-cytoplasmic trafficking of proteins play a key role in the development of leukemia and drug resistance. KPT-330 (Selinexor), an inhibitor of chromosome region maintenance 1 (CRM1, nuclear receptor exportin 1, XPO1), demonstrated activities against a few hematological malignancies. We examined the anti-leukemic efficacy of KPT-330 in IM-resistant CML. Cell viability was examined by MTS assay. Apoptosis and cell cycle were assessed by flow cytometry. CRM1 mRNA was detected by PCR. Expression of CRM1 protein and its cargo proteins were determined by western blot or immunofluorescent staining. Furthermore, we engrafted nude mice subcutaneously with IM-resistant CML K562G. Mice were treated with IM, KPT-330 alone or in combination. Expression of CRM1 in CML were markedly higher than control. KPT-330 inhibited proliferation, induced cell cycle arrest and apoptosis of K562 and K562G. IC50 of IM on K562G was reduced by KPT-330. Mechanistically, KPT-330 inhibited CRM1 and increased the nuclear/cytoplasm ratio of BCR-ABL and P27. p-AKT was downregulated while p-STAT1 and caspase-3 were upregulated. Furthermore, KPT-330 showed anti-leukemic effect in primary IM-resistant CML with T315I mutation in CRM1-dependent manner. In K562G xenograft mice model, KPT-330 inhibited tumor growth and sensitized K562G to IM in vivo. To conclude, KPT-330 showed anti-leukemic activity and sensitized CML to IM in CRM1-dependent manner in vitro and in vivo. KPT-330 represents an alternative therapy for IM-refractory CML, warranting further investigation of CRM1 as therapeutic target.

CRM1, a novel independent prognostic factor overexpressed in invasive breast carcinoma of poor prognosis.

Breast cancer (BC) is the most commonly diagnosed cancer in females globally and is more aggressive at later stages. Chromosome region maintenance 1 (CRM1) is involved in the nuclear export of proteins and RNAs and has been associated with a number of malignancies. However, the clinicopathological significance of its expression in BC remains to be elucidated therefore this was investigated in the present study. CRM1 expression in 280 breast cancer tissues and 60 normal tissues was retrospectively analyzed using immunohistochemistry (IHC) and western blotting. IHC investigation demonstrated that CRM1 expression was significantly increased in BC compared with the normal breast epithelium (P<0.0001). Overexpression of CRM1 was markedly associated with poor prognostic characteristics, including larger tumor size (P=0.024), positive lymph node metastasis (P=0.032), invasive histological type (P=0.004) and distant metastasis (P=0.026). Significant associations were also observed between increased CRM1 expression and the progesterone receptor (P=0.028) and Ki67 (P=0.019). Kaplan-Meier survival analysis demonstrated that patients with high CRM1 expression exhibited a reduced disease-free survival and overall survival compared with those with low CRM1 expression (P=0.013). In the multivariate analysis, CRM1 expression (P=0.011), tumor size (P=0.001) and lymph node metastasis (P<0.001) were independent prognostic markers of BC. In conclusion, CRM1 serves an important role in BC and may serve as a predictive and prognostic factor for a poor outcome in patients with BC.

Antagonism of Integrin CD11b Affords Protection against Endotoxin Shock and Polymicrobial Sepsis via Attenuation of HMGB1 Nucleocytoplasmic Translocation and Extracellular Release.

High mobility group box 1 (HMGB1), a chromatin-binding nuclear protein, plays a critical role in sepsis by acting as a key "late-phase" inflammatory mediator. Integrin CD11b is essential for inflammatory cell activation and migration, thus mediating inflammatory responses. However, it is unclear whether CD11b participates in the development of sepsis. In this study, we report that CD11b contributes to LPS-induced endotoxin shock and microbial sepsis, as antagonism of CD11b with the CD11b blocking Ab or CD11b inhibitor Gu-4 protects mice against LPS- and microbial sepsis-related lethality, which is associated with significantly diminished serum HMGB1 levels. Consistent with this, CD11b-deficient mice were more resistant to microbial sepsis with a much lower serum HMGB1 level compared with wild-type mice. Pharmacological blockage and genetic knockdown/knockout of CD11b in murine macrophages hampered LPS-stimulated HMGB1 nucleocytoplasmic translocation and extracellular release. Furthermore, silencing CD11b interrupted the interaction of HMGB1 with either a nuclear export factor chromosome region maintenance 1 or classical protein kinase C and inhibited classical protein kinase C-induced HMGB1 phosphorylation, the potential underlying mechanism(s) responsible for CD11b blockage-induced suppression of HMGB1 nucleocytoplasmic translocation and subsequent extracellular release. Thus, our results highlight that CD11b contributes to the development of sepsis, predominantly by facilitating nucleocytoplasmic translocation and active release of HMGB1.

Regulation of inflammatory responses by dynamic subcellular localization of RNA-binding protein Arid5a

Adenine-thymine (AT)-rich interactive domain 5a (Arid5a) is an RNA-binding protein found in the cytoplasm and nucleus of normally growing cells. Although Arid5a is known to play an important role in immune regulation, whether and how Arid5a subcellular localization impacts immune regulation has remained unclear. In this study, we generated Arid5a transgenic (TG) mice to address this question. While ectopic Arid5a overexpression did not affect expression of inflammatory cytokines under unstimulated conditions, significantly higher levels of inflammatory cytokines, such as IL-6, were produced in response to lipopolysaccharide (LPS) stimulation. Consistent with this, TG mice were more sensitive to LPS treatment than wild-type mice. We also found that Arid5a is imported into the nucleus via a classical importin-α/β1-mediated pathway. On stimulation, nuclear Arid5a levels were decreased, while there was a concomitant increase in cytoplasmic Arid5a. Arid5a is associated with up-frameshift protein 1, and its nuclear export is regulated by a nuclear export receptor, chromosomal region maintenance 1. Taken together, these data indicate that Arid5a is a dynamic protein that translocates to the cytoplasm from the nucleus so as to properly exert its dual function in mRNA stabilization and transcriptional regulation during inflammatory conditions.

Verdinexor Targeting of CRM1 is a Promising Therapeutic Approach against RSV and Influenza Viruses.

Two primary causes of respiratory tract infections are respiratory syncytial virus (RSV) and influenza viruses, both of which remain major public health concerns. There are a limited number of antiviral drugs available for the treatment of RSV and influenza, each having limited effectiveness and each driving selective pressure for the emergence of drug-resistant viruses. Novel broad-spectrum antivirals are needed to circumvent problems with current disease intervention strategies, while improving the cytokine-induced immunopathology associated with RSV and influenza infections. In this review, we examine the use of Verdinexor (KPT-335, a novel orally bioavailable drug that functions as a selective inhibitor of nuclear export, SINE), as an antiviral with multifaceted therapeutic potential. KPT-335 works to (1) block CRM1 (i.e., Chromosome Region Maintenance 1; exportin 1 or XPO1) mediated export of viral proteins critical for RSV and influenza pathogenesis; and (2) repress nuclear factor κB (NF-κB) activation, thus reducing cytokine production and eliminating virus-associated immunopathology. The repurposing of SINE compounds as antivirals shows promise not only against RSV and influenza virus but also against other viruses that exploit the nucleus as part of their viral life cycle.

Does Size Really Matter? Probing the Efficacy of Structural Reduction in the Optimization of Bioderived Compounds – A Computational “Proof-of-Concept”

Over the years, numerous synthetic approaches have been utilized in drug design to improve the pharmacological properties of naturally derived compounds and most importantly, minimize toxic effects associated with their transition to drugs. The reduction of complex bioderived compounds to simpler bioactive fragments has been identified as a viable strategy to develop lead compounds with improved activities and minimal toxicities. Although this ‘reductive’ strategy has been widely exemplified, underlying biological events remain unresolved, hence the unanswered question remains how does the fragmentation of a natural compound improve its bioactivity and reduce toxicities? Herein, using a combinatorial approach, we initialize a computational “proof-of- concept” to expound the differential pharmacological and antagonistic activities of a natural compound, Anguinomycin D, and its synthetic fragment, SB640 towards Exportin Chromosome Region Maintenance 1 (CRM1). Interestingly, our findings revealed that in comparison with the parent compound, SB640 exhibited improved pharmacological attributes, while toxicities and off-target activities were relatively minimal. Moreover, we observed that the reduced size of SB640 allowed ‘deep access’ at the Nuclear Export Signals (NES) binding groove of CRM1, which favored optimal and proximal positioning towards crucial residues while the presence of the long polyketide tail in Anguinomycin D constrained its burial at the hydrophobic groove. Furthermore, with regards to their antagonistic functions, structural inactivation (rigidity) was more pronounced in CRM1 when bound by SB640 as compared to Anguinomycin D. These findings provide essential insights that portray synthetic fragmentation of natural compounds as a feasible approach towards the discovery of potential leads in disease treatment.

Characterization of the Nucleocytoplasmic Transport Mechanisms of Epstein-Barr Virus BFLF2

Background/Aims: Epstein-Barr virus (EBV) BFLF2, the homologue of herpes simplex virus 1 (HSV-1) UL31, is crucial for the efficient viral DNA packaging and primary egress across the nuclear membrane. However, we still do not know its subcellular transport mechanisms. Methods: Interspecies heterokaryon assays were utilized to detect the nucleocytoplasmic shuttling of BFLF2, and mutation analysis, plasmid transfection and fluorescence microscopy assays were performed to identify the functional nuclear localization sequence (NLS) and nuclear export sequence (NES) of BFLF2 in live cells. Furthermore, the nuclear import and export of BFLF2 were assessed by confocal microscopy, co-immunoprecipitation and immunoblot assays. Results: BFLF2 was confirmed to shuttle between the nucleus and cytoplasm. Two predicted NESs were shown to be nonfunctional, yet we proved that the nuclear export of BFLF2 was mediated through transporter associated with antigen processing (TAP), but not chromosomal region maintenance 1 (CRM1) dependent pathway. Furthermore, one functional NLS, ²²RRLMHPHHRNYTASKASAH⁴⁰, was identified, and the aa22-23, aa22-25, aa28-30 and aa37-40 had an important role in the nuclear localization of BFLF2. Besides, the nuclear import of BFLF2 was demonstrated through Ran-, importin α7-, importin β1- and transportin-1-dependent mechanism that does not require importin α1, α3 and α5. Conclusion: These works are of significance for the further study of the functions of BFLF2 during EBV infection, as well as for further insights into the design of new antiviral drug target and vaccine development against EBV.

Selinexor in Combination with Cladribine, Cytarabine and G-CSF for Relapsed or Refractory AML

Selinexor is an oral, reversible, potent Selective Inhibitor of Nuclear Export / SINETM compound that specifically blocks the protein Exportin 1 (XPO1), also known as chromosomal region maintenance 1 (CRM1). Selinexor induces cell cycle arrest, inhibits DNA damage repair and modulates the expression of NF-κB in cancer cells lines and has demonstrated broad activity across several hematologic malignancies including AML. Our group has previously demonstrated that selinexor restores sensitivity to both cytarabine and anthracycline resistant AML cell lines. Furthermore, combination therapy with selinexor and cytarabine showed additive effects on inhibition of cell proliferation in vitro and prolonged the survival of leukemic mice in vivo . (Rettig et al., ASH 2013). For these reasons, we tested the safety and efficacy of selinexor in combination with chemotherapy in patients with relapsed or refractory AML.Eligible patients were between 18-70 years of age with AML and either a) primary refractory disease following ≤ 2 cycles of induction chemotherapy, b) first relapse with no prior unsuccessful salvage chemotherapy, or c) relapsed or refractory to hypomethylating agents. Selinexor 60 mg/d was administered on days 1, 5, 10, and 12 in combination with CLAG, (cladribine 5 mg/m2/d on d4-8, cytarabine 2000 mg/m2/d on d4-8 and G-CSF 300 mcg/d on d3-8). The primary endpoint was rate of complete remission (CR + CRi) for selinexor + CLAG per 2003 IWG criteria.Thirty patients with a median age of 56 years (range 21-70) have been enrolled in the study of which 19 (63%) were treated in first relapse, 10 (33%) failed induction chemotherapy and 1 (3%) was refractory to hypomethylating agent. The complete remission rate for 28 evaluable patients was 50%, (7 CR, 7 CRi). Median time to neutrophil recovery (ANC >1,000/mm3) following treatment was 28 days (range 21-52). Toxicity data is available for the first 27 patients. The most common nonhematologic treatment emergent adverse events included weight loss (n=17, 63%) nausea (16, 59%), mucositis (16, 59%), elevations in AST (15, 56%), ALT (13, 48%), alk phos (13, 48%), and fatigue (13, 48%), the majority of which were G1-2. Serious adverse events occurred in 14 patients, the most frequently due to sepsis (n=6, 22%). Early death within 30 days of the start of treatment occurred only in 1 subject (4%) due to respiratory failure. Of the initial 25 patients treated, 15 have undergone subsequent allogeneic hematopoietic cell transplantation (60%). We conclude that selinexor + CLAG is highly active in patients with relapsed or refractory AML and has encouraging rates of CR. Furthermore, the combination serves as a bridge which allows a high percentage of patients to undergo allogeneic hematopoietic cell transplantation. DisclosuresUy:GlycoMimetics: Consultancy; Novartis: Consultancy, Other: Travel Suppport; Boehringer Ingelheim: Consultancy. Riedell:Novartis: Membership on an entity's Board of Directors or advisory committees; Kite Pharma: Speakers Bureau. Vij:Amgen: Honoraria, Research Funding; Jazz: Honoraria; Konypharma: Honoraria; Takeda: Honoraria, Research Funding; Bristol-Meyers-Squibb: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Abbvie: Honoraria.

SUMOylation regulates nuclear accumulation and signaling activity of the soluble intracellular domain of the ErbB4 receptor tyrosine kinase

Erb-B2 receptor tyrosine kinase 4 (ErbB4) is a kinase that can signal via a proteolytically released intracellular domain (ICD) in addition to classical receptor tyrosine kinase-activated signaling cascades. Previously, we have demonstrated that ErbB4 ICD is posttranslationally modified by the small ubiquitin-like modifier (SUMO) and functionally interacts with the PIAS3 SUMO E3 ligase. However, direct evidence of SUMO modification in ErbB4 signaling has remained elusive. Here, we report that the conserved lysine residue 714 in the ErbB4 ICD undergoes SUMO modification, which was reversed by sentrin-specific proteases (SENPs) 1, 2, and 5. Although ErbB4 kinase activity was not necessary for the SUMOylation, the SUMOylated ErbB4 ICD was tyrosine phosphorylated to a higher extent than unmodified ErbB4 ICD. Mutation of the SUMOylation site compromised neither ErbB4-induced phosphorylation of the canonical signaling pathway effectors Erk1/2, Akt, or STAT5 nor ErbB4 stability. In contrast, SUMOylation was required for nuclear accumulation of the ErbB4 ICD. We also found that Lys-714 was located within a leucine-rich stretch, which resembles a nuclear export signal, and could be inactivated by site-directed mutagenesis. Furthermore, SUMOylation modulated the interaction of ErbB4 with chromosomal region maintenance 1 (CRM1), the major nuclear export receptor for proteins. Finally, the SUMO acceptor lysine was functionally required for ErbB4 ICD-mediated inhibition of mammary epithelial cell differentiation in a three-dimensional cell culture model. Our findings indicate that a SUMOylation-mediated mechanism regulates nuclear localization and function of the ICD of ErbB4 receptor tyrosine kinase.

Pharmacological treatment with inhibitors of nuclear export enhances the antitumor activity of docetaxel in human prostate cancer.

Background and aimsDocetaxel (DTX) modestly increases patient survival of metastatic castration-resistant prostate cancer (mCRPC) due to insurgence of pharmacological resistance. Deregulation of Chromosome Region Maintenance (CRM-1)/ exportin-1 (XPO-1)-mediated nuclear export may play a crucial role in this phenomenon.Material and methodsHere, we evaluated the effects of two Selective Inhibitor of Nuclear Export (SINE) compounds, selinexor (KPT-330) and KPT-251, in association with DTX by using 22rv1, PC3 and DU145 cell lines with their. DTX resistant derivatives.Results and conclusionsWe show that DTX resistance may involve overexpression of β-III tubulin (TUBB3) and P-glycoprotein as well as increased cytoplasmic accumulation of Foxo3a. Increased levels of XPO-1 were also observed in DTX resistant cells suggesting that SINE compounds may modulate DTX effectiveness in sensitive cells as well as restore the sensitivity to DTX in resistant ones. Pretreatment with SINE compounds, indeed, sensitized to DTX through increased tumor shrinkage and apoptosis by preventing DTX-induced cell cycle arrest. Basally SINE compounds induce FOXO3a activation and nuclear accumulation increasing the expression of FOXO-responsive genes including p21, p27 and Bim causing cell cycle arrest. SINE compounds-catenin and survivin supporting apoptosis. βdown-regulated Cyclin D1, c-myc, Nuclear sequestration of p-Foxo3a was able to reduce ABCB1 and TUBB3 H2AX levels, prolonged γ expression. Selinexor treatment increased DTX-mediated double strand breaks (DSB), and reduced the levels of DNA repairing proteins including DNA PKc and Topo2A. Our results provide supportive evidence for the therapeutic use of SINE compounds in combination with DTX suggesting their clinical use in mCRPC patients.

Suppression of nucleocytoplasmic p27Kip1 export attenuates CDK4-mediated neuronal death induced by status epilepticus

Aberrant cell cycle re-entry promotes neuronal death in various neurological diseases. Thus, cyclin-dependent kinases (CDKs) seem to be one of potential therapeutic targets to prevent neuronal loss. In the present study, we investigated the involvements of CDK4, CDK5 and p27Kip1 (an endogenous CDK inhibitor) in status epilepticus (SE)-induced neuronal death. Following SE, CDK4 expression was increased in CA1 neurons, while CDK5 was decreased. Most of TUNEL-positive neurons showed CDK4 expression, but less CDK5 expression. Flavopiridol (a CDK4 inhibitor) attenuated TUNEL signal and CDK4 expression in CA1 neurons following SE. CDK5 inhibitors did not affect these phenomena. Both flavopiridol and leptomycin B (an inhibitor of chromosome region maintenance 1) mitigated SE-induced neuronal death by inhibiting nucleocytoplasmic p27Kip1 translocation. These findings suggest that SE may lead to nucleocytoplasmic p27Kip1 export that initiates CDK4, not CDK5, induction, which an abortive and fatal cell cycle re-entry progress in CA1 neurons.

Leptomycin B reduces primary and acquired resistance of gefitinib in lung cancer cells

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) gefitinib has demonstrated dramatic clinical efficacy in non-small cell lung cancer (NSCLC) patients. However, its therapeutic efficacy is ultimately limited by the development of acquired drug resistance. The aim of this study was to explore the potential utility of chromosome region maintenance 1 (CRM1) inhibitor leptomycin B (LMB) in combination with gefitinib to overcome primary and acquired gefitinib resistance in NSCLC cells. The combinative effects of gefitinib and LMB were evaluated by MTT and its underlining mechanism was assessed by flow cytometry and Western blot. LMB displayed a synergistic effect on gefitinib-induced cytotoxicity in A549 (IC50: 25.0±2.1μM of gefitinib+LMB vs. 32.0±2.5μM of gefitinib alone, p<0.05). Gefitinib+LMB caused a significantly different cell cycle distribution and signaling pathways involved in EGFR/survivin/p21 compared with gefitinib. A549 cells then were treated with progressively increased concentrations of gefitinib (A549GR) or in combination with LMB (A549GLR) over 10months to generate gefitinib resistance. IC50 of gefitinib in A549GLR (37.0±2.8μM) was significantly lower than that in A549GR (53.0±3.0μM, p<0.05), which indicates that LMB could reverse gefitinib-induced resistance in A549. Further mechanism investigation revealed that the expression patterns of EGFR pathway and epithelial-mesenchymal transition (EMT) markers in A549, A549GR, and A549GLR were significantly different. In conclusion, LMB at a very low concentration (0.5nM) combined with gefitinib showed synergistic therapeutic effects and ameliorated the development of gefitinib-induced resistance in lung cancer cells.

TFIIA transcriptional activity is controlled by a 'cleave-and-run' Exportin-1/Taspase 1-switch.

Transcription factor TFIIA is controlled by complex regulatory networks including proteolysis by the protease Taspase 1, though the full impact of cleavage remains elusive. Here, we demonstrate that in contrast to the general assumption, de novo produced TFIIA is rapidly confined to the cytoplasm via an evolutionary conserved nuclear export signal (NES, amino acids 21VINDVRDIFL30), interacting with the nuclear export receptor Exportin-1/chromosomal region maintenance 1 (Crm1). Chemical export inhibition or genetic inactivation of the NES not only promotes TFIIA's nuclear localization but also affects its transcriptional activity. Notably, Taspase 1 processing promotes TFIIA's nuclear accumulation by NES masking, and modulates its transcriptional activity. Moreover, TFIIA complex formation with the TATA box binding protein (TBP) is cooperatively enhanced by inhibition of proteolysis and nuclear export, leading to an increase of the cell cycle inhibitor p16INK, which is counteracted by prevention of TBP binding. We here identified a novel mechanism how proteolysis and nuclear transport cooperatively fine-tune transcriptional programs.