Silencing of KPNA2 inhibits high glucose-induced podocyte injury via inactivation of mTORC1/p70S6K signaling pathway

Dysregulation of apoptotic and autophagic function are characterized as the main pathogeneses of diabetic nephropathy (DN). It has been reported that Karyopherin Alpha 2 (KPNA2) contributes to apoptosis and autophagy in various cells, but its role in DN development remains unknown. The purpose of present study was to explore the function and underling mechanisms of KPNA2 in development of DN. In this study, 30 mM high glucose (HG)-evoked podocytes were used as DN model. The expression of KPNA2 was detected by qRT-PCR and Western blot assays. The cell viability was tested by CCK-8 kit, the apoptosis was measured using flow cytometry assay, the apoptotic and the autophagy related genes was detected by Western blot. Our results indicated that KPNA2 was significantly increased after HG stimulation. Knockdown of KPNA2 inhibited apoptosis, and promoted cell viability and autophagy in HG-treated podocytes. In addition, silencing of KPNA2 deactivated mTORC1/p70S6K pathway activation via regulating SLC1A5. Further results demonstrated that activating mTORC1/p70S6K pathway strongly ameliorated the effect of KPNA2 on cell viability, apoptosis and autophagy. Therefore, our study suggested that knockdown of KPNA2 rescued HG-induced injury via blocking activation of mTORC1/p70S6K pathway by mediating SLC1A5.     

NPM1 Silencing Reduces Tumour Growth and MAPK Signalling in Prostate Cancer Cells

The chaperone nucleophosmin (NPM1) is over-expressed in the epithelial compartment of prostate tumours compared to adjacent healthy epithelium and may represent one of the key actors that support the neoplastic phenotype of prostate adenocarcinoma cells. Yet, the mechanisms that underlie NPM1 mediated phenotype remain elusive in the prostate. To better understand NPM1 functions in prostate cancer cells, we sought to characterize its impact on prostate cancer cells behaviour and decipher the mechanisms by which it may act. Here we show that NPM1 favors prostate tumour cell migration, invasion and colony forming. Furthermore, knockdown of NPM1 leads to a decrease in the growth of LNCaP-derived tumours grafted in Nude mice in vivo. Such oncogenic-like properties are found in conjunction with a positive regulation of NPM1 on the ERK1/2 (Extracellular signal-Regulated Kinases 1/2) kinase phosphorylation in response to EGF (Epidermal Growth Factor) stimulus, which is critical for prostate cancer progression following the setting of an autonomous production of the growth factor. NPM1 could then be a target to switch off specifically ERK1/2 pathway activation in order to decrease or inhibit cancer cell growth and migration.     

Nucleophosmin is required for DNA integrity and p19Arf protein stability

Nucleophosmin (NPM) is a nucleolar phosphoprotein that binds the tumor suppressors p53 and p19(Arf) and is thought to be indispensable for ribogenesis, cell proliferation, and survival after DNA damage. The NPM gene is the most frequent target of genetic alterations in leukemias and lymphomas, though its role in tumorigenesis is unknown. We report here the first characterization of a mouse NPM knockout strain. Lack of NPM expression results in accumulation of DNA damage, activation of p53, widespread apoptosis, and mid-stage embryonic lethality. Fibroblasts explanted from null embryos fail to grow and rapidly acquire a senescent phenotype. Transfer of the NPM mutation into a p53-null background rescued apoptosis in vivo and fibroblast proliferation in vitro. Cells null for both p53 and NPM grow faster than control cells and are more susceptible to transformation by activated oncogenes, such as mutated Ras or overexpressed Myc. In the absence of NPM, Arf protein is excluded from nucleoli and is markedly less stable. Our data demonstrate that NPM regulates DNA integrity and, through Arf, inhibits cell proliferation and are consistent with a putative tumor-suppressive function of NPM.     

Distinct roles for classical nuclear import receptors in the growth of multinucleated muscle cells

Proper muscle function is dependent on spatial and temporal control of gene expression in myofibers. Myofibers are multinucleated cells that are formed, repaired and maintained by the process of myogenesis in which progenitor myoblasts proliferate, differentiate and fuse. Gene expression is dependent upon proteins that require facilitated nuclear import, however little is known about the regulation of nucleocytoplasmic transport during the formation of myofibers. We analyzed the role of karyopherin alpha (KPNA), a key classical nuclear import receptor, during myogenesis. We established that five karyopherin alpha paralogs are expressed by primary mouse myoblasts in vitro and that their steady-state levels increase in multinucleated myotubes, suggesting a global increase in demand for classical nuclear import during myogenesis. We used siRNA-mediated knockdown to identify paralog-specific roles for KPNA1 and KPNA2 during myogenesis. KPNA1 knockdown increased myoblast proliferation, whereas KPNA2 knockdown decreased proliferation. In contrast, no proliferation defect was observed with KPNA4 knockdown. Only knockdown of KPNA2 decreased myotube growth. These results identify distinct pathways involved in myoblast proliferation and myotube growth that rely on specific nuclear import receptors suggesting that regulation of classical nuclear import pathways likely plays a critical role in controlling gene expression in skeletal muscle.     

Wip1 cooperates with KPNA2 to modulate the cell proliferation and migration of colorectal cancer via a p53-dependent manner

Due to the increasing incidence and mortality, the early diagnosis, specific targeted therapies, and prognosis for colorectal cancer (CRC) attract more and more attention. Wild-type p53-induced phosphatase 1 (Wip1) and karyopherin α2 (KPNA2) have been regarded as oncogenes in many cancers, including CRC. Wip1 dephosphorylates p53 to inactivate it. TP53 activator and Wip1 inhibitor downregulate KPNA2 expression. Therefore, we speculate that Wip1 may co-operate with KPNA2 to modulate CRC progression in a p53-dependent manner. Here, Wip1 and KPNA2 messenger RNA expression and protein levels are significantly increased in CRC tissues and cell lines and are positively correlated with each other. Wip1 silence increases p53 phosphorylation while decreases KPNA2 protein. Wip1 knockdown remarkably suppresses CRC cell proliferation and migration while KPNA2 overexpression exerts an opposing effect. KPNA2 overexpression could partially rescue Wip1 silence-inhibited CRC cell proliferation and migration. Finally, Wip1 interacts with KPNA2 to modulate the activation of AKT/GSK-3β signaling and metastasis-related factors. In summary, Wip1 could co-operate with KPNA2 to modulate CRC cell proliferation and migration, possibly via a p53-dependent manner, through downstream AKT/GSK-3β pathway. We provided a novel mechanism of Wip1 interacting with KPNA2, therefore modulating CRC cell proliferation and migration.     

GLTSCR2 is an upstream negative regulator of nucleophosmin in cervical cancer

Nucleophosmin (NPM)/B23, a multifunctional nucleolar phosphoprotein, plays an important role in ribosome biogenesis, cell cycle regulation, apoptosis and cancer pathogenesis. The role of NPM in cells is determined by several factors, including total expression level, oligomerization or phosphorylation status, and subcellular localization. In the nucleolus, NPM participates in rRNA maturation to enhance ribosomal biogenesis. Consistent with this finding, NPM expression is increased in rapidly proliferating cells and many types of human cancers. In response to ribosomal stress, NPM is redistributed to the nucleoplasm, where it inactivates mouse double minute 2 homologue to stabilize p53 and inhibit cell cycle progression. These observations indicate that nucleolus‐nucleoplasmic mobilization of NPM is one of the key molecular mechanisms that determine the role of NPM within the cell. However, the regulatory molecule(s) that control(s) NPM stability and subcellular localization, crucial to the pluripotency of intercellular NPM, remain(s) unidentified. In this study, we showed that nucleolar protein GLTSCR2/Pict‐1 induced nucleoplasmic translocation and enhanced the degradation of NPM via the proteasomal polyubiquitination pathway. In addition, we showed that GLTSCR2 expression decreased the transforming activity of cells mediated by NPM and that the expression of NPM is reciprocally related to that of GLTSCR2 in cervical cancer tissue. In this study, we demonstrated that GLTSCR2 is an upstream negative regulator of NPM.     

Important role of the LKB1-AMPK pathway in suppressing tumorigenesis in PTEN-deficient mice

The LKB1 tumour suppressor phosphorylates and activates AMPK (AMP-activated protein kinase) when cellular energy levels are low, thereby suppressing growth through multiple pathways, including inhibiting the mTORC1 (mammalian target of rapamycin complex 1) kinase that is activated in the majority of human cancers. Blood glucose-lowering Type 2 diabetes drugs also induce LKB1 to activate AMPK, indicating that these compounds could be used to suppress growth of tumour cells. In the present study, we investigated the importance of the LKB1-AMPK pathway in regulating tumorigenesis in mice resulting from deficiency of the PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumour suppressor, which drives cell growth through overactivation of the Akt and mTOR (mammalian target of rapamycin) kinases. We demonstrate that inhibition of AMPK resulting from a hypomorphic mutation that decreases LKB1 expression does not lead to tumorigenesis on its own, but markedly accelerates tumour development in PTEN(+/-) mice. In contrast, activating the AMPK pathway by administration of metformin, phenformin or A-769662 to PTEN(+/-) mice significantly delayed tumour onset. We demonstrate that LKB1 is required for activators of AMPK to inhibit mTORC1 signalling as well as cell growth in PTEN-deficient cells. Our findings highlight, using an animal model relevant to understanding human cancer, the vital role that the LKB1-AMPK pathway plays in suppressing tumorigenesis resulting from loss of the PTEN tumour suppressor. They also suggest that pharmacological inhibition of LKB1 and/or AMPK would be undesirable, at least for the treatment of cancers in which the mTORC1 pathway is activated. Most importantly, our results demonstrate the potential of AMPK activators, such as clinically approved metformin, as anticancer agents, which will suppress tumour development by triggering a physiological signalling pathway that potently inhibits cell growth.     

Long noncoding RNA ATB promotes ovarian cancer tumorigenesis by mediating histone H3 lysine 27 trimethylation through binding to EZH2

Ovarian cancer (OC) remains one of the most lethal gynecological malignancies. The unfavourable prognosis is mainly due to the lack of early-stage diagnosis, drug resistance and recurrence. Therefore, it needs to investigate the mechanism of OC tumorigenesis and identify effective biomarkers for the clinical diagnosis. It is reported that long noncoding RNAs (lncRNAs) play important roles during the tumorigenesis of OC. Therefore, the present study aimed to study the role and clinical significance of LncRNAs ATB (lnc-ATB) in the development and progression of OC. In our research, lnc-ATB expression in OC tissues was elevated compared with adjacent normal tissues and high expression of lnc-ATB was associated with poor outcomes of OC patients. The silencing of lnc-ATB blocked cell proliferation, invasion and migration in SKOV3 and A2780 cells. RNA immunoprecipitation and RNA pull-down results showed that lnc-ATB positively regulated the expression of EZH2 via directly interacting with EZH2. Besides, the overexpression of EZH2 partly rescued lnc-ATB silencing-inducing inhibition of cell proliferation, invasion and migration. Chromatin immunoprecipitation assay results demonstrated that the silencing of lnc-ATB reduced the occupancy of caudal-related homeobox protein 1, Forkhead box C1, Large tumour suppressor kinase 2, cadherin-1 and disabled homolog 2 interacting protein promoters on EZH2 and H3K27me3. These data revealed the oncogenic of lnc-ATB and provided a novel biomarker for OC diagnosis. Furthermore, these findings indicated the mechanism of lnc-ATB functioning in the progression of OC, which provided a new target for OC therapy.     

The germacranolide sesquiterpene lactone neurolenin B of the medicinal plant Neurolaena lobata (L.) R.Br. ex Cass inhibits NPM/ALK-driven cell expansion and NF-κB-driven tumour intravasation

BackgroundThe t(2;5)(p23;q35) chromosomal translocation results in the expression of the fusion protein NPM/ALK that when expressed in T-lymphocytes gives rise to anaplastic large cell lymphomas (ALCL). In search of new therapy options the dichloromethane extract of the ethnomedicinal plant Neurolaena lobata (L.) R.Br. ex Cass was shown to inhibit NPM/ALK expression.PurposeTherefore, we analysed whether the active principles that were recently isolated and found to inhibit inflammatory responses specifically inhibit growth of NPM/ALK+ ALCL, leukaemia and breast cancer cells, but not of normal cells, and the intravasation through the lymphendothelial barrier.MethodsALCL, leukaemia and breast cancer cells, and normal peripheral blood mononuclear cells (PBMCs) were treated with isolated sesquiterpene lactones and analysed for cell cycle progression, proliferation, mitochondrial activity, apoptosis, protein and mRNA expression, NF-κB and cytochrome P450 activity, 12(S)-HETE production and lymphendothelial intravasation.ResultsIn vitro treatment of ALCL by neurolenin B suppressed NPM/ALK, JunB and PDGF-Rβ expression, inhibited the growth of ALCL cells late in M phase, and induced apoptosis via caspase 3 without compromising mitochondrial activity (as a measure of general exogenic toxicity). Moreover, neurolenin B attenuated tumour spheroid intravasation probably through inhibition of NF-κB and CYP1A1.ConclusionNeurolenin B specifically decreased pro-carcinogenic NPM/ALK expression in ALK+ ALCL cells and, via the inhibition of NF-kB signalling, attenuated tumour intra/extravasation into the lymphatics. Hence, neurolenin B may open new options to treat ALCL and to manage early metastatic processes to which no other therapies exist.     

BIG3 Inhibits the Estrogen-Dependent Nuclear Translocation of PHB2 via Multiple Karyopherin-Alpha Proteins in Breast Cancer Cells

We recently reported that brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3) binds Prohibitin 2 (PHB2) in cytoplasm, thereby causing a loss of function of the PHB2 tumor suppressor in the nuclei of breast cancer cells. However, little is known regarding the mechanism by which BIG3 inhibits the nuclear translocation of PHB2 into breast cancer cells. Here, we report that BIG3 blocks the estrogen (E2)-dependent nuclear import of PHB2 via the karyopherin alpha (KPNA) family in breast cancer cells. We found that overexpressed PHB2 interacted with KPNA1, KPNA5, and KPNA6, thereby leading to the E2-dependent translocation of PHB2 into the nuclei of breast cancer cells. More importantly, knockdown of each endogenous KPNA by siRNA caused a significant inhibition of E2-dependent translocation of PHB2 in BIG3-depleted breast cancer cells, thereby enhancing activation of estrogen receptor alpha (ERα). These data indicated that BIG3 may block the KPNAs (KPNA1, KPNA5, and KPNA6) binding region(s) of PHB2, thereby leading to inhibition of KPNAs-mediated PHB2 nuclear translocation in the presence of E2 in breast cancer cells. Understanding this regulation of PHB2 nuclear import may provide therapeutic strategies for controlling E2/ERα signals in breast cancer cells.     

Epigenetic silencing of the WNT antagonist Dickkopf 3 disrupts normal Wnt/β‐catenin signalling and apoptosis regulation in breast cancer cells

Dickkopf‐related protein 3 (DKK3) is an antagonist of Wnt ligand activity. Reduced DKK3 expression has been reported in various types of cancers, but its functions and related molecular mechanisms in breast tumorigenesis remain unclear. We examined the expression and promoter methylation of DKK3 in 10 breast cancer cell lines, 96 primary breast tumours, 43 paired surgical margin tissues and 16 normal breast tissues. DKK3 was frequently silenced in breast cell lines (5/10) by promoter methylation, compared with human normal mammary epithelial cells and tissues. DKK3 methylation was detected in 78% of breast tumour samples, whereas only rarely methylated in normal breast and surgical margin tissues, suggesting tumour‐specific methylation of DKK3 in breast cancer. Ectopic expression of DKK3 suppressed cell colony formation through inducing G0/G1 cell cycle arrest and apoptosis of breast tumour cells. DKK3 also induced changes of cell morphology, and inhibited breast tumour cell migration through reversing epithelial‐mesenchymal transition (EMT) and down‐regulating stem cell markers. DKK3 inhibited canonical Wnt/β‐catenin signalling through mediating β‐catenin translocation from nucleus to cytoplasm and membrane, along with reduced active‐β‐catenin, further activating non‐canonical JNK signalling. Thus, our findings demonstrate that DKK3 could function as a tumour suppressor through inducing apoptosis and regulating Wnt signalling during breast tumorigenesis.