Oridonin inhibits the occurrence and development of colorectal cancer by reversing the Warburg effect via reducing PKM2 dimer formation and preventing its entry into the nucleus.

BACKGROUND: The Warburg effect is prevalent in human cancer. Oridonin (ORI) has excellent anticancer effects, but its exact anticancer mechanism is still unclear. METHODS: CCK8, EdU, and flow cytometry assay were performed to detect the effect of ORI on cell viability, proliferation and apoptosis, respectively. RNA-seq was carried out to search the underlying mechanisms. Total PKM2, dimeric PKM2, nuclear PKM2 was detected by Western blot. The epidermal growth factor receptor/extracellular signal regulated kinase (EGFR/ERK) signaling was assayed. The binding ability of Importin-α5 to PKM2 was performed by Co-IP experiments. The effect of ORI combined with cysteine (Cys) or fructose-1, 6-diphosphate (FDP) on cancer cells was detected. Mouse xenograft model was established to confirm the molecular mechanisms in vivo. RESULTS: ORI inhibited viability, proliferation and promoted apoptosis of CRC cells. RNA-seq revealed ORI attenuated the Warburg effect in cancer cells. ORI reduced dimeric PKM2 and prevented it from entering the nucleus. ORI did not affect the EGFR/ERK signaling, but reduced Importin-α5 binding to the PKM2 dimer. Cys or FDP reversed or enhanced the effect of ORI. Animal model assay confirmed the molecular mechanisms in vivo. CONCLUSIONS: Our study first shows that ORI could have anticancer activity by inhibiting the Warburg effect as a novel activator of PKM2.

Dual targeting of protein translation and nuclear protein export results in enhanced antimyeloma effects.

Selinexor (KPT-330) is a small molecule inhibitor of XPO1, which mediates the transport of tumor suppressor proteins, oncogene messenger RNAs, and other proteins involved in governing cell growthfrom the cell nucleus to the cytoplasm. It is overexpressed in many cancer types. Because eukaryotic translation initiator factor 4E (eIF4E) plays a critical role in protein translation in cancer cells in multiple myeloma (MM), we evaluated the effectiveness of combined inhibition of protein translation and nuclear export in MM. Selinexor, an inhibitor of nuclear protein export, dose-dependently decreased eIF4E, IKZF1, and c-MYC protein levels. Using a doxycycline-inducible-pLKO-Tet-On vector, knockdown of eIF4E significantly enhanced the antiproliferative effects of selinexor, sensitized resistant MM cells to selinexor, and increased apoptosis in MM cells. Immunofluorescent analysis of MM cells showed that the combined treatment increased the localization of residual eIF4E to the nucleus compared with selinexor-only treatment. The overexpression of eIF4E at least partially rescued the effects of selinexor in MM cells by reducing G1 cell cycle arrest and increasing the selinexor-IC50 10-fold. Moreover, the combination of selinexor with pharmacologic inhibitors of protein translation showed synergistic anti-MM effects. These results suggest a synergistic anti-MM effect of selinexor combined with eIF4E inhibitors in vitro. Our work provides a better understanding of the potential mechanism of resistance to selinexor and a rationale for combining selinexor with eIF4E inhibitors for the treatment of MM.

Reactive Oxygen Species-Induced Inhibition of Odontoblastic Differentiation of Mouse Dental Papilla Cells Is Mediated by Downregulation of Importin 7.

Tooth dentin is a crucial tooth structure. The biological process of odontoblast differentiation is essential for formation of normal dentin. Accumulation of reactive oxygen species (ROS) leads to oxidative stress, which can influence the differentiation of several cells. As a member of the importin-ß superfamily, importin 7 (IPO7) is essential for nucleocytoplasmic transport and plays an important role in the processes of odontoblast differentiation and oxidative stress. Nevertheless, the association between ROS, IPO7, and odontoblast differentiation in mouse dental papilla cells (mDPCs) and the underlying mechanisms remain to be elucidated. In this study, we confirmed that ROS suppressed odontoblastic differentiation of mDPCs as well as the expression and nucleocytoplasmic shuttle of IPO7 in cells, while overexpression of IPO7 can rescue these effects. ROS resulted in increased phosphorylation of p38 and cytoplasmic aggregation of phosphorylated p38 (p-p38), which was able to be reversed by overexpression of IPO7. p-p38 interacted with IPO7 in mDPCs without hydrogen peroxide (H2O2) treatment, but in the presence of H2O2, the interaction between p-p38 and IPO7 was significantly decreased. Inhibition of IPO7 increased the expression level and nuclear translocation of p53, which are mediated by cytoplasmic aggregation of p-p38. In conclusion, ROS inhibited odontoblastic differentiation of mDPCs, which is mediated by downregulation and damaged nucleocytoplasmic shuttle of IPO7.

Inhibition of XPO1 impairs cholangiocarcinoma cell proliferation by triggering p53 intranuclear accumulation.

BACKGROUND: XPO1 mediates the nuclear export of several proteins, mainly tumor suppressors. KPT-330 (Selinexor) is a selective inhibitor of XPO1 that has demonstrated good therapeutic effects in hematologic cancers. METHODS: We used TCGA and GTEx pan-cancer database to evaluate XPO1 mRNA expression in various tumors. Cell proliferation assay and colony formation assay were used to analyze the in vitro antitumor effects of XPO1 inhibitor KPT-330. Western blot was performed to explore the specific mechanisms. RESULTS: We found that XPO1 was highly expressed across a range of cancers and associated with poor prognosis in hepatobiliary and pancreatic tumors. We revealed that the XPO1 inhibitor KPT-330 triggered the nuclear accumulation of the p53 protein and significantly disrupted the proliferation of cholangiocarcinoma cells. Mechanistically, the XPO1 inhibitor, KPT-330, reduced BIRC6 expression by inhibiting the PI3K/AKT pathway to decrease p53 degradation and improve its stability. CONCLUSION: Therefore, XPO1 may be a potential therapeutic target in cholangiocarcinoma, mediated by its effects on KPT-330.

A highly potent small-molecule antagonist of exportin-1 selectively eliminates CD44+CD24- enriched breast cancer stem-like cells.

Breast cancer stem-like cells (BCSCs) have been suggested as the underlying cause of tumor recurrence, metastasis and drug resistance in triple-negative breast cancer (TNBC). Here, we report the discovery and biological evaluation of a highly potent small-molecule antagonist of exportin-1, LFS-1107. We ascertained that exportin-1 (also named as CRM1) is a main cellular target of LFS-1107 by nuclear export functional assay, bio-layer interferometry binding assay and C528S mutant cell line. We found that LFS-1107 significantly inhibited TNBC tumor cells at low-range nanomolar concentration and LFS-1107 can selectively eliminate CD44+CD24- enriched BCSCs. We demonstrated that LFS-1107 can induce the nuclear retention of Survivin and consequent strong suppression of STAT3 transactivation abilities and the expression of downstream stemness regulators. Administration of LFS-1107 can strongly inhibit tumor growth in mouse xenograft model and eradicate BCSCs in residual tumor tissues. Moreover, LFS-1107 can significantly ablate the patient-derived tumor organoids (PDTOs) of TNBC as compared to a few approved cancer drugs. Lastly, we revealed that LFS-1107 can enhance the killing effects of chemotherapy drugs and downregulate multidrug resistance related protein targets. These new findings provide preclinical evidence of defining LFS-1107 as a promising therapeutic agent to deplete BCSCs for the treatment of TNBC.

The E3 ubiquitin ligase CHIP protects against sepsis-induced myocardial dysfunction by inhibiting NF-κB-mediated inflammation via promoting ubiquitination and degradation of karyopherin-α 2.

Cardiac dysfunction has been recognized as a major contributor to mortality in sepsis, which is closely associated with inflammatory reactions. The carboxy terminus of Hsc70-interacting protein (CHIP), a U-box E3 ubiquitin ligase, defends against cardiac injury caused by other factors, but its role in sepsis-induced cardiac dysfunction has yet to be determined. The present study was designed to investigate the effects of CHIP on cardiac dysfunction caused by sepsis and the molecular mechanisms underlying these processes. We discovered that the CHIP level decreased gradually in the heart at different time points after septic model construction. The decline in CHIP expression of lipopolysaccharide (LPS)-stimulated cardiomyocytes was related to c-Jun activation that inhibited the transcription of CHIP. Functional biology experiments indicated that CHIP bound directly to karyopherin-α 2 (KPNA2) and promoted its degradation through polyubiquitination in cardiomyocytes. CHIP overexpression in cardiomyocytes obviously inhibited LPS-initiated release of TNF-α and IL-6 by promoting KPNA2 degradation, reducing NF-κB translocation into the nucleus. Consistent with the in vitro results, data obtained from animal experiments indicated that septic transgenic mice with heart-specific CHIP overexpression showed a weaker proinflammatory response and reduced cardiac dysfunction than septic control mice. Furthermore, we found that the therapeutic effect of compound YL-109 on cardiac dysfunction in septic mice was due to the upregulation of myocardial CHIP expression. These findings demonstrated that sepsis-initiated the activation of c-Jun suppressed CHIP transcription. CHIP directly promoted ubiquitin-mediated degradation of KPNA2, which reduced the production of proinflammatory cytokines by inhibiting the translocation of NF-κB from the cytoplasm into the nucleus in myocardium, thereby attenuating sepsis-induced cardiac dysfunction.

Discovery of Highly Potent Daphnane Diterpenoids Uncovers Importin-ß1 as a Druggable Vulnerability in Castration-Resistant Prostate Cancer.

Importins are overexpressed in many cancers and mediate the abnormal nuclear transport of oncogenic factors. The druggable potential of importins still remains unclear, largely because of the lack of potent inhibitors. Herein, the anti-castration-resistant prostate cancer (CRPC) screening of a Euphorbiaceae diterpenoid library followed by target fishing led to the identification of a highly potent importin-ß1 inhibitor, daphnane diterpenoid DD1. DD1 selectively inhibited the growth and survival of CRPC cells at subnanomolar concentrations and completely blocked tumor growth in preclinical models at an extremely low dosage. Mechanistic studies revealed that targeting of importin-ß1 by DD1 significantly reduced the nuclear accumulation of key CRPC drivers, shutting down their downstream oncogenic signaling. Disruption of the predicted binding sites of DD1 on importin-ß1 abolished this anti-CRPC effect. These findings suggest that importin-ß1 is an effective therapeutic target in CRPC and that DD1 as the most potent importin-ß1 inhibitor to date can be developed as therapeutics for treatment of this disease.

Long noncoding RNA SNHG14 knockdown exerts a neuroprotective role in MPP+-induced Parkinson's disease cell model through mediating miR-135b-5p/KPNA4 axis.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disease resulted from the loss of dopaminergic neurons. Here, we analyzed the role of long noncoding RNA (lncRNA) small nucleolar RNA host gene 14 (SNHG14) in PD using 1-methyl-4-phenyl pyridine (MPP+)-induced PD cell model. METHODS: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot assay were performed to determine RNA and protein expression, respectively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry (FCM) analysis were conducted to analyze cell viability and apoptosis. Enzyme-Linked Immunosorbent Assay (ELISA) was conducted to analyze the release of inflammatory cytokines. Cytotoxicity was assessed using reactive oxygen species (ROS) assay kit, superoxide dismutase (SOD) activity assay kit and lactate dehydrogenase (LDH) activity assay kit. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were performed to confirm the interaction between microRNA-135b-5p (miR-135b-5p) and SNHG14 or karyopherin subunit alpha 4 (KPNA4). RESULTS: MPP+ treatment elevated the expression of SNHG14 in SK-N-SH cells in a dose and time-dependent manner. SNHG14 knockdown alleviated MPP+-induced apoptosis, inflammation, and cytotoxicity in SK-N-SH cells. SNHG14 interacted with miR-135b-5p, and SNHG14 silencing-mediated effects were partly overturned by miR-135b-5p knockdown in PD cell model. Besides, miR-135b-5p interacted with the 3' untranslated region (3'UTR) of KPNA4, and KPNA4 overexpression partly reversed miR-135b-5p overexpression-induced effects in PD cell model. SNHG14 knockdown reduced the protein level of KPNA4 partly by up-regulating miR-135b-5p in SK-N-SH cells. CONCLUSION: SNHG14 promoted MPP+-induced neuro injury in PD cell model through mediating miR-135b-5p/KPNA4 axis.

Exportin 1 Inhibition Induces Nerve Growth Factor Receptor Expression to Inhibit the NF-κB Pathway in Preclinical Models of Pediatric High-Grade Glioma.

High-grade glioma (HGG) is the leading cause of cancer-related death among children. Selinexor, an orally bioavailable, reversible inhibitor of the nuclear export protein, exportin 1, is in clinical trials for a range of cancers, including HGG. It inhibits the NF-κB pathway and strongly induces the expression of nerve growth factor receptor (NGFR) in preclinical cancer models. We hypothesized that selinexor inhibits NF-κB via upregulation of NGFR. In HGG cells, sensitivity to selinexor correlated with increased induction of cell surface NGFR expression. Knocking down NGFR in HGG cells increased proliferation, anchorage-independent growth, stemness markers, and levels of transcriptionally available nuclear NF-κB not bound to IκB-α, while decreasing apoptosis and sensitivity to selinexor. Increasing IκB-α levels in NGFR knockdown cells restored sensitivity to selinexor. Overexpression of NGFR using cDNA reduced levels of free nuclear NF-κB, decreased stemness markers, and increased markers of cellular differentiation. In all HGG lines tested, selinexor decreased phosphorylation of NF-κB at serine 536 (a site associated with increased transcription of proliferative and inflammatory genes). Because resistance to selinexor monotherapy occurred in our in vivo model, we screened selinexor with a panel of FDA-approved anticancer agents. Bortezomib, a proteasome inhibitor that inhibits the NF-κB pathway through a different mechanism than selinexor, showed synergy with selinexor against HGG in vitro Our results help elucidate selinexor's mechanism of action and identify NGFR as a potential biomarker of its effect in HGG and in addition suggest a combination therapy strategy for these challenging tumors.

CRM1-mediated nuclear export of dengue virus RNA polymerase NS5 modulates interleukin-8 induction and virus production.

Although all established functions of dengue virus NS5 (nonstructural protein 5) occur in the cytoplasm, its nuclear localization, mediated by dual nuclear localization sequences, is essential for virus replication. Here, we have determined the mechanism by which NS5 can localize in the cytoplasm to perform its role in replication, establishing for the first time that it is able to be exported from the nucleus by the exportin CRM1 and hence can shuttle between the nucleus and cytoplasm. We define the nuclear export sequence responsible to be residues 327-343 and confirm interaction of NS5 and CRM1 by pulldown assay. Significantly, greater nuclear accumulation of NS5 during infection due to CRM1 inhibition coincided with altered kinetics of virus production and decreased induction of the antiviral chemokine interleukin-8. This is the first report of a nuclear export sequence within NS5 for any member of the Flavivirus genus; because of its high conservation within the genus, it may represent a target for the treatment of diseases caused by several medically important flaviviruses.

Inhibition of the CRM1-mediated nucleocytoplasmic transport by N-azolylacrylates: structure-activity relationship and mechanism of action.

CRM1-mediated nucleocytoplasmic transport plays an important role in many cellular processes and diseases. To investigate the structural basis required for the inhibition of the CRM1-mediated nuclear export we have synthesized analogs of a previously identified small molecule lead compound and monitored their activity against the Rev function of the human immunodeficiency virus. Microscopy studies show that the active congeners of this series inhibit the nucleocytoplasmic transport of Rev and the co-localization between Rev and CRM1 in living cells. Mechanism of action studies show their interaction with the Cys528 residue of CRM1 involving a Michael-addition type of reaction. However, structure-activity relationship demonstrates strict constraints to the structure of the inhibitors, and shows that activity is not solely correlated to Michael-addition suggesting a more complex mechanism of action. Our results are suggestive for the existence of a well-defined interaction at the CRM1-NES binding site. In addition, the most selective congener inhibited the HIV-1 production in latently infected cells. These specific CRM1 inhibitors are of interest as tool for analyzing the mechanisms of post-transcriptional control of gene expression and provide insight in the design of new agents.

CRM1-dependent nuclear export of dengue virus type 2 NS5.

The dengue virus multidomain RNA polymerase NS5 has been observed in the nucleus in mammalian infected cell systems. We previously showed that NS5 nuclear localization is mediated by two nuclear targeting signals within the NS5 interdomain region that are recognized by distinct members of the importin superfamily of intracellular transporters. Intriguingly, we have recently found that NS5 also possesses the ability to be exported from the nucleus by the importin family member CRM1 (exportin 1) both in Vero cells transfected to express NS5, and in dengue virus type 2 infected Vero cells, based on use of the CRM1-specific inhibitor leptomycin B (LMB). LMB treatment of Vero cells resulted in increased nuclear accumulation in both systems, and interestingly in the latter, resulted in an alteration in the kinetics of virus production. Our results imply that subcellular trafficking of NS5 at particular times in the infectious cycle may be central to the kinetics of virus production; perturbing this trafficking may represent a viable approach to develop new antiviral therapeutics.

Nuclear localization of glyceraldehyde-3-phosphate dehydrogenase is not involved in the initiation of apoptosis induced by 1-Methyl-4-phenyl-pyridium iodide (MPP+).

Nuclear localization of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is implicated in the process of apoptosis. To study the function of GAPDH, we expressed GAPDH C-terminally fused with or without nuclear localization signal (NLS) in SH-SY5Y and NB41A3 cells using a retrovirus expression system. GAPDH carrying NLS (GAPDH-NLS) was expressed mainly in the nucleus. However, expression of GAPDH-NLS did not cause any difference in cell survival rate as compared to that of the vector alone or GAPDH without NLS. Treatment with 1-Methyl-4-phenyl-pyridium iodide (MPP+) caused no difference in the cell survival rate or in the pattern or extent of apoptosis among the three transductants. In the cells expressing GAPDH without NLS, MPP+ did not cause visible translocation of GAPDH into nucleus before the onset of apoptosis. Since GAPDH is known to comprise a CRM1-mediated nuclear export signal, we blocked the nuclear export of GAPDH by treatment with leptomycin B, an inhibitor of CRM1-mediated nuclear export. The treatment did not cause any difference in apoptosis among the three transductants. An additional treatment with MPP+ induced no apoptotic difference in these cells. Thus, we have concluded that a simple nuclear localization of GAPDH does not induce apoptosis, and that MPP+-induced apoptosis is not caused by nuclear translocation of GAPDH.

The CRM1 nuclear export receptor controls pathological cardiac gene expression.

Diverse pathological insults trigger a cardiac remodeling process during which myocytes undergo hypertrophy, with consequent decline in cardiac function and eventual heart failure. Multiple transcriptional regulators of pathological cardiac hypertrophy are controlled at the level of subcellular distribution. For example, prohypertrophic transcription factors belonging to the nuclear factor of activated T cells (NFAT) and GATA families are subject to CRM1-dependent nuclear export but are rapidly relocalized to the nucleus in response to cues for hypertrophic growth. Here, we demonstrate that the antihypertrophic chromatin-modifying enzyme histone deacetylase 5 (HDAC5) is shuttled out of the cardiomyocyte nucleus via a CRM1-mediated pathway in response to diverse signals for hypertrophy. CRM1 antagonists block the agonist-mediated nuclear export of HDAC 5 and repress pathological gene expression and associated hypertrophy of cultured cardiomyocytes. Conversely, CRM1 activity is dispensable for nonpathological cardiac gene activation mediated by thyroid hormone and insulin-like growth factor 1, agonists that fail to trigger the nuclear export of HDAC5. These results suggest a selective role for CRM1 in derepression of pathological cardiac genes via its neutralizing effects on antihypertrophic factors such as HDAC5. Pharmacological approaches targeting CRM1-dependent nuclear export in heart muscle may have salutary effects on cardiac function by suppressing maladaptive changes in gene expression evoked by stress signals.