Nucleoporin-93 overexpression overcomes multiple nucleocytoplsamic trafficking bottlenecks to permit robust metastasis

Abstract To identify genetic events driving breast cancer progression, we performed multi-omics integrated analyses that identified overexpression of nucleoporin-93, a nuclear pore component. Here we show that NUP93 overexpression enhances trans-endothelial migration and matrix invasion in vitro, along with metastasis in animal models. These findings were supported by analyses of naturally occurring activating NUP93 mutations and inactivating nephrotic syndrome mutations. Mechanistically, NUP93 activation enhanced the ultimate nuclear transport step shared by multiple signaling pathways, including TGF-beta/SMAD, EGF/ERK and TNF/NF-κB. Likewise, NUP93 can boost nuclear transport of beta-catenin, as well as elevate expression and inhibit degradation of this co-activator. The emerging addiction to nuclear transport exposes vulnerabilities of tumors overexpressing NUP93. Congruently, we report that myristoylated peptides corresponding to the nuclear translocation signals of SMAD and ERK inhibited growth and metastasis. Our study illuminates a previously unappreciated hallmark of advanced tumors, which derive benefit from unblocking a set of nucleocytoplasmic bottlenecks.

Download Full-text

266 Effects of Inflammatory States on Ovarian Biology and Oocyte Competence

Journal of Animal Science ◽

10.1093/jas/skab235.246 ◽

2021 ◽

Vol 99 (Supplement_3) ◽

pp. 134-135

Author(s):

Jennifer A Hernandez Gifford ◽

Emily Ferranti ◽

Kylee Forrest ◽

Craig A Gifford

Keyword(s):

Granulosa Cell ◽

Oocyte Maturation ◽

Beta Catenin ◽

Control Mechanisms ◽

Steroid Production ◽

Oocyte Competence ◽

Nuclear Maturation ◽

Naturally Occurring ◽

Hormonal Milieu

Abstract Female fertility is dependent on estradiol concentrations which regulate a multitude of ovarian functions including follicle development and oocyte maturation leading to ovulation of a viable oocyte. Estradiol biosynthesis is regulated by coordinated actions of follicle-stimulating hormone and intra-ovarian control mechanisms including the co-transcription factor beta-catenin. Beta-catenin is a multi-faceted protein recognized for its role in granulosa cell steroid production and is shown to be modulated by lipopolysaccharide (LPS), the endotoxin responsible for stimulation of the immune system in infections caused by Gram-negative bacteria. Beef heifers treated with subacute concentrations of LPS during a synchronized follicular wave demonstrate a decline in serum estradiol concentrations 50 h after CIDR withdrawal, corresponding with dominant follicle maturation and preceding ovulation. The endotoxin exposure also resulted in increased LPS concentration and E2:P4 ratios in follicular fluid suggesting that low dose LPS modulates the intra-follicular hormonal milieu. Additionally, in a granulosa cell line, LPS treatment decreased mRNA expression of aromatase and beta-catenin. These data indicate that LPS alters E2 synthesis by modulating beta-catenin and subsequent steroidogenic enzyme expression. To further explore the contribution of naturally occurring LPS exposure on follicular steroid production and developing oocytes, bovine ovary pairs were collected from local abattoirs. Oocytes were aspirated from small follicles and matured in vitro to evaluate meiotic events related to nuclear maturation and spindle morphology. Small follicles from ovarian pairs were separated by the detectable LPS concentrations into high and low LPS groups. Oocytes matured from low LPS follicles demonstrated an increase in the percent of abnormal maturation events. Data indicate that induced or naturally occurring low doses of LPS can alter circulating and follicular estradiol concentrations impairing oocyte maturation. Perturbation to local ovarian signaling cascades from subclinical inflammatory disease may be an unappreciated factor altering fertility and leading to decreased cow retention.

Download Full-text

Identification of four nuclear transport signal-binding proteins that interact with diverse transport signals

Molecular and Cellular Biology ◽

10.1128/mcb.9.7.3028-3036.1989 ◽

1989 ◽

Vol 9 (7) ◽

pp. 3028-3036

Author(s):

L Yamasaki ◽

P Kanda ◽

R E Lanford

Keyword(s):

Binding Proteins ◽

Nuclear Transport ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Nuclear Pore ◽

Signal Peptides ◽

Signal Recognition ◽

Transport Pathway

The transport of proteins into the nucleus requires not only the presence of a nuclear transport signal on the targeted protein but also the signal recognition proteins and the nuclear pore translocation apparatus. Complicating the search for the signal recognition proteins is the fact that the nuclear transport signals identified share little obvious homology. In this study, synthetic peptides homologous to the nuclear transport signals from the simian virus 40 large T antigen, Xenopus oocyte nucleoplasmin, adenovirus E1A, and Saccharomyces cerevisiae MAT alpha 2 proteins were coupled to a UV-photoactivable cross-linker and iodinated for use in an in vitro cross-linking reaction with cellular lysates. Four proteins, p140, p100, p70, and p55, which specifically interacted with the nuclear transport signal peptides were identified. Unique patterns of reactivity were observed with closely related pairs of nuclear transport signal peptides. Competition experiments with labeled and unlabeled peptides demonstrated that heterologous signals were able to bind the same protein and suggested that diverse signals use a common transport pathway. The subcellular distribution of the four nuclear transport signal-binding proteins suggested that nuclear transport involves both cytoplasmic and nuclear receptors. The four proteins were not bound by wheat germ agglutinin and were not associated tightly with the nuclear pore complex.

Download Full-text

Mevalonate Pathway Enzyme HMGCS1 Contributes to Gastric Cancer Progression

Cancers ◽

10.3390/cancers12051088 ◽

2020 ◽

Vol 12 (5) ◽

pp. 1088 ◽

Cited By ~ 2

Author(s):

I-Han Wang ◽

Tzu-Ting Huang ◽

Ji-Lin Chen ◽

Li-Wei Chu ◽

Yueh-Hsin Ping ◽

...

Keyword(s):

Gastric Cancer ◽

Endoplasmic Reticulum ◽

Cancer Cells ◽

Cancer Progression ◽

Nuclear Translocation ◽

Mevalonate Pathway ◽

Gastric Cancer Cells ◽

Gastric Cancer Progression

The 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1) is a potential regulatory node in the mevalonate pathway that is frequently dysregulated in tumors. This study found that HMGCS1 expression is upregulated in stomach adenocarcinoma samples of patients and tumorspheres of gastric cancer cells. HMGCS1 elevates the expression levels of the pluripotency genes Oct4 and SOX-2 and contributes to tumorsphere formation ability in gastric cancer cells. HMGCS1 also promotes in vitro cell growth and progression and the in vivo tumor growth and lung metastasis of gastric cancer cells. After blocking the mevalonate pathway by statin and dipyridamole, HMGCS1 exerts nonmetabolic functions in enhancing gastric cancer progression. Furthermore, the level and nuclear translocation of HMGCS1 in gastric cancer cells are induced by serum deprivation. HMGCS1 binds to and activates Oct4 and SOX-2 promoters. HMGCS1 also enhances the integrated stress response (ISR) and interacts with the endoplasmic reticulum (ER) stress transducer protein kinase RNA-like endoplasmic reticulum kinase (PERK). Our results reveal that HMGCS1 contributes to gastric cancer progression in both metabolic and nonmetabolic manners.

Download Full-text

Monoclonal Antibodies to NTF2 Inhibit Nuclear Protein Import by Preventing Nuclear Translocation of the GTPase Ran

Molecular Biology of the Cell ◽

10.1091/mbc.11.2.703 ◽

2000 ◽

Vol 11 (2) ◽

pp. 703-719 ◽

Cited By ~ 26

Author(s):

Susanne M. Steggerda ◽

Ben E. Black ◽

Bryce M. Paschal

Keyword(s):

Nuclear Pore Complex ◽

Nuclear Import ◽

Protein Import ◽

Nuclear Translocation ◽

Living Cells ◽

Nuclear Accumulation ◽

Nuclear Pore ◽

Permeabilized Cells ◽

Dependent Protein

Nuclear transport factor 2 (NTF2) is a soluble transport protein originally identified by its ability to stimulate nuclear localization signal (NLS)-dependent protein import in digitonin-permeabilized cells. NTF2 has been shown to bind nuclear pore complex proteins and the GDP form of Ran in vitro. Recently, it has been reported that NTF2 can stimulate the accumulation of Ran in digitonin-permeabilized cells. Evidence that NTF2 directly mediates Ran import or that NTF2 is required to maintain the nuclear concentration of Ran in living cells has not been obtained. Here we show that cytoplasmic injection of anti-NTF2 mAbs resulted in a dramatic relocalization of Ran to the cytoplasm. This provides the first evidence that NTF2 regulates the distribution of Ran in vivo. Moreover, anti-NTF2 mAbs inhibited nuclear import of both Ran and NLS-containing protein in vitro, suggesting that NTF2 stimulates NLS-dependent protein import by driving the nuclear accumulation of Ran. We also show that biotinylated NTF2-streptavidin microinjected into the cytoplasm accumulated at the nuclear envelope, indicating that NTF2 can target a binding partner to the nuclear pore complex. Taken together, our data show that NTF2 is an essential regulator of the Ran distribution in living cells and that NTF2-mediated Ran nuclear import is required for NLS-dependent protein import.

Download Full-text

A non-functional copy of the salmonid sex determining gene (sdY) is responsible for the “apparent” XY females in Chinook salmon, Oncorhynchus tshawytscha

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkab451 ◽

2022 ◽

Author(s):

Sylvain Bertho ◽

Amaury Herpin ◽

Elodie Jouanno ◽

Ayaka Yano ◽

Julien Bobe ◽

...

Keyword(s):

Missense Mutation ◽

Chinook Salmon ◽

Oncorhynchus Tshawytscha ◽

Nuclear Translocation ◽

Naturally Occurring ◽

Determination System ◽

Synergistic Induction ◽

Sex Determination System

Abstract Many salmonids have a male heterogametic (XX/XY) sex determination system, and they are supposed to have a conserved master sex determining gene (sdY), that interacts at the protein level with Foxl2 leading to the blockage of the synergistic induction of Foxl2 and Nr5a1 of the cyp19a1a promoter. However, this hypothesis of a conserved master sex determining role of sdY in salmonids is challenged by a few exceptions, one of them being the presence of naturally occurring “apparent” XY Chinook salmon, Oncorhynchus tshawytscha, females. Here we show that some XY Chinook salmon females have a sdY gene (sdY-N183), with one missense mutation leading to a substitution of a conserved isoleucine to an asparagine (I183N). In contrast, Chinook salmon males have both a non-mutated sdY-I183 gene and the missense mutation sdY-N183 gene. The 3D model of SdY-I183N predicts that the I183N hydrophobic to hydrophilic amino acid change leads to a modification of the SdY β-sandwich structure. Using in vitro cell transfection assays we found that SdY-I183N, like the wildtype SdY, is preferentially localized in the cytoplasm. However, compared to wildtype SdY, SdY-I183N is more prone to degradation, its nuclear translocation by Foxl2 is reduced and SdY-I183N is unable to significantly repress the synergistic Foxl2/Nr5a1 induction of the cyp19a1a promoter. Altogether our results suggest that the sdY-N183 gene of XY Chinook females is non-functional and that SdY-I183N is no longer able to promote testicular differentiation by impairing the synthesis of estrogens in the early differentiating gonads of wild Chinook salmon XY females.

Download Full-text

Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores.

The Journal of Cell Biology ◽

10.1083/jcb.104.2.189 ◽

1987 ◽

Vol 104 (2) ◽

pp. 189-200 ◽

Cited By ~ 295

Author(s):

D R Finlay ◽

D D Newmeyer ◽

T M Price ◽

D J Forbes

Keyword(s):

Rat Liver ◽

Protein Transport ◽

Nuclear Protein ◽

Nuclear Transport ◽

Nuclear Pore ◽

Major Mechanism ◽

Rat Liver Nuclei ◽

Egg Extract ◽

Liver Nuclei

Selective transport of proteins is a major mechanism by which biochemical differences are maintained between the cytoplasm and nucleus. To begin to investigate the molecular mechanism of nuclear transport, we used an in vitro transport system composed of a Xenopus egg extract, rat liver nuclei, and a fluorescently labeled nuclear protein, nucleoplasmin. With this system, we screened for inhibitors of transport. We found that the lectin, wheat germ agglutinin (WGA), completely inhibits the nuclear transport of fluorescently labeled nucleoplasmin. No other lectin tested affected nuclear transport. The inhibition by WGA was not seen when N-acetylglucosamine was present and was reversible by subsequent addition of sugar. When rat liver nuclei that had been incubated with ferritin-labeled WGA were examined by electron microscopy, multiple molecules of WGA were found bound to the cytoplasmic face of each nuclear pore. Gel electrophoresis and nitrocellulose transfer identified one major and several minor nuclear protein bands as binding 125I-labeled WGA. The most abundant protein of these, a 63-65-kD glycoprotein, is a candidate for the inhibitory site of action of WGA on nuclear protein transport. WGA is the first identified inhibitor of nuclear protein transport and interacts directly with the nuclear pore.

Download Full-text

Arctigenin Attenuates Breast Cancer Progression through Decreasing GM-CSF/TSLP/STAT3/β-Catenin Signaling

International Journal of Molecular Sciences ◽

10.3390/ijms21176357 ◽

2020 ◽

Vol 21 (17) ◽

pp. 6357

Author(s):

Hui Shi ◽

Luping Zhao ◽

Xinlin Guo ◽

Runping Fang ◽

Hui Zhang ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Cancer Progression ◽

Breast Cancer Cells ◽

Nuclear Translocation ◽

Breast Cancer Progression ◽

Gm Csf ◽

Stat3 Phosphorylation

Invasive breast cancer is highly regulated by tumor-derived cytokines in tumor microenvironment. The development of drugs that specifically target cytokines are promising in breast cancer treatment. In this study, we reported that arctigenin, a bioactive compound from Arctium lappa L., could decrease tumor-promoting cytokines GM-CSF, MMP-3, MMP-9 and TSLP in breast cancer cells. Arctigenin not only inhibited the proliferation, but also the invasion and stemness of breast cancer cells via decreasing GM-CSF and TSLP. Mechanistically, arctigenin decreased the promoter activities of GM-CSF and TSLP via reducing the nuclear translocation of NF-κB p65 which is crucial for the transcription of GM-CSF and TSLP. Furthermore, arctigenin-induced depletion of GM-CSF and TSLP inhibited STAT3 phosphorylation and β-catenin signaling resulting in decreased proliferation, invasion and stemness of breast cancer cells in vitro and in vivo. Our findings provide new insights into the mechanism by which tumor-promoting cytokines regulate breast cancer progression and suggest that arctigenin is a promising candidate for cytokine-targeted breast cancer therapy.

Download Full-text

The Novel Thienopyrimidine Derivative, RP-010, Produces Its Efficacy in Prostate Cancer Cells by Inducing the Fragmentation of β-catenin

10.20944/preprints201903.0045.v1 ◽

2019 ◽

Author(s):

Haneen Amawi ◽

Noor Hussein ◽

Sai HS Boddu ◽

Chandrabose Karthikeyan ◽

Frederick E. Williams ◽

...

Keyword(s):

Prostate Cancer ◽

Cancer Cells ◽

Signaling Pathway ◽

Nuclear Translocation ◽

Biologically Active ◽

Beta Catenin ◽

Lead Compound ◽

Prostate Cancer Cells

Thienopyrimidines are a versatile group of compounds that contain a biologically active pharmacophore and reported to have anticancer efficacy in vitro. Here, we report for the first time, that thieno[3,2-d]pyrimidine - based compounds, designated the RP series, have efficacy in prostate cancer cells. The lead compound, RP-010, was efficacious in PC3 and DU-145 prostate cancer (PC) cells (IC50< 1µM). The cytotoxicity of RP-010 was significantly lower in normal cells. RP-010 (0.5, 1, 2, and 4 µM) arrested prostate cancer cells in the G2 phase of the cell cycle, induced mitotic catastrophe and apoptotic signaling in both PC cell lines. Mechanistic studies suggested that RP-010 (1 and 2 µM) inhibits the wingless-type MMTV (Wnt)/β-catenin signaling pathway, mainly by inducing β-catenin fragmentation, while down regulating important proteins in the pathway, i.e. LRP-6, DVL3, and c-Myc. Interestingly, RP-010 (1 and 2 µM) induced the nuclear translocation of the negative feedback proteins, Naked 1 and Naked 2, in the signaling pathway. In addition, RP-010 (0.5, 1, 2, and 4 µM) significantly decreased the migration and invasiveness of PC cells in vitro. Finally, RP-010 did not produce significant toxic effects in zebrafish at concentrations up to 6 µM. In conclusion, RP-10 is a promising anticancer compound in metastatic prostate cancer and did not produce overt toxicity in an in vivo zebrafish model. Future mechanistic and efficacy studies are needed in-vivo to optimize the lead compound RP-010 for clinical use.

Download Full-text

From the clinic to the bench and back again in one dog year: identifying new treatments for sarcoma using a cross-species personalized medicine pipeline

10.1101/517086 ◽

2019 ◽

Author(s):

Sneha Rao ◽

Jason A. Somarelli ◽

Erdem Altunel ◽

Laura E. Selmic ◽

Mark Byrum ◽

...

Keyword(s):

Personalized Medicine ◽

Exome Sequencing ◽

Cancer Progression ◽

Initial Response ◽

Driver Mutations ◽

Cancer Drug ◽

Naturally Occurring ◽

New Treatments

AbstractCancer drug discovery is an inefficient process, with more than 90% of newly-discovered therapies failing to gain regulatory approval. Patient-derived models of cancer offer a promising new approach to identifying personalized treatments; however, for rare cancers, such as sarcomas, access to patient samples can be extremely limited, which precludes development of patient-derived models. To address the limited access to patient samples, we have turned to pet dogs with naturally-occurring sarcomas. Although sarcomas make up less than 1% of all cancers in humans, sarcomas represent at least 15% of all cancers in dogs. Dogs with naturally-occurring sarcomas also have intact immune systems, an accelerated pace of cancer progression, and share the same environment as humans, making them ideal models that bridge key gaps between mouse models and human sarcomas.Here, we develop a framework for a personalized medicine pipeline that integrates drug screening, validation, and genomics to identify new therapies. We tested this paradigm through the study of a pet dog, Teddy, who presented with six synchronous leiomyosarcomas. By integrating patient-derived cancer models, in vitro drug screens, and in vivo validation we identified proteasome inhibitors as a potential therapy for Teddy. After showing an initial response to the proteasome inhibitor, bortezomib, Teddy developed rapid resistance, and tumor growth resumed. Whole exome sequencing revealed substantial genetic heterogeneity across Teddy’s multiple recurrent tumors and metastases, suggesting that intra-patient heterogeneity was responsible for the heterogeneous clinical response. Ubiquitin proteomics coupled with exome sequencing revealed multiple candidate driver mutations in proteins related to the proteasome pathway. Together, our results demonstrate how the comparative study of canine sarcomas can offer rapid insights into the process of developing personalized medicine approaches that can lead to new treatments for sarcomas in both humans and canines.

Download Full-text

PECAM-1 (CD31) functions as a reservoir for and a modulator of tyrosine-phosphorylated beta-catenin

Journal of Cell Science ◽

10.1242/jcs.112.18.3005 ◽

1999 ◽

Vol 112 (18) ◽

pp. 3005-3014 ◽

Cited By ~ 2

Author(s):

N. Ilan ◽

S. Mahooti ◽

D.L. Rimm ◽

J.A. Madri

Keyword(s):

Endothelial Cell ◽

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Nuclear Translocation ◽

Adherens Junction ◽

Cell Contact ◽

Beta Catenin ◽

Dependent Manner

Catenins function as regulators of cellular signaling events in addition to their previously documented roles in adherens junction formation and function. Evidence to date suggests that beta and gamma catenins can act as signaling molecules, bind transcriptional factors and translocate to the nucleus. Beta- and gamma-catenin are also major substrates for protein tyrosine kinases, and tyrosine phosphorylation of junctional proteins is correlated with decreased adhesiveness. One way in which catenin functions are modulated is by dynamic incorporation into junctional complexes which controls, in part, the cytoplasmic levels of catenins. Here we show that: (1) vascular endothelial growth factor (VEGF) induces beta-catenin tyrosine phosphorylation in a time-, and dose-dependent manner and that VEGF receptors co-localize to areas of endothelial cell-cell contact in vitro and in vivo. (2) Platelet-endothelial cell adhesion molecule (PECAM)-1 can function as a reservoir for, and modulator of, tyrosine phosphorylated beta-catenin. (3) PECAM-1 can prevent beta-catenin nuclear translocation in transfected SW480 colon carcinoma cells. We suggest that PECAM-1 may play a role in modulating beta-catenin tyrosine phosphorylation levels, localization and signaling and by doing so, functions as an important modulator of the endothelium.

Download Full-text