scholarly journals A role for the autophagic receptor, SQSTM1/p62, in trafficking NF-κB/RelA to nucleolar aggresomes

2020 ◽  
Author(s):  
Ian T Lobb ◽  
Pierre Morin ◽  
Kirsty Martin ◽  
Xhordi Lieshi ◽  
Karl Olsen ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Cancer Progression ◽  
Dominant Negative ◽  
Regulatory Proteins ◽  
Dominant Negative Mutant ◽  
Contributory Factor ◽  
Hallmarks Of Cancer ◽  
Solid Malignancies ◽  
Elevated Activity ◽  
Related Proteins

AbstractElevated NF-κB activity is a contributory factor in many haematological and solid malignancies. Nucleolar sequestration of NF-κB/RelA represses this elevated activity and mediates apoptosis of cancer cells. Here we set out to understand the mechanisms that control the nuclear/nucleolar distribution of RelA and other regulatory proteins, so that agents can be developed that specifically target these proteins to the organelle. We demonstrate that RelA accumulates in intra-nucleolar aggresomes in response to specific stresses. We also demonstrate that the autophagy receptor, SQSTM1/p62, accumulates alongside RelA in these nucleolar aggresomes. This accumulation is not a consequence of inhibited autophagy. Indeed, our data suggest nucleolar and autophagosomal accumulation of p62 are in active competition. We identify a conserved motif at the N-terminus of p62 that is essential for nucleoplasmic-to nucleolar transport of the protein. Furthermore, using a dominant negative mutant deleted for this nucleolar localisation signal (NoLS), we demonstrate a role for p62 in trafficking RelA and other aggresome-related proteins to nucleoli. Together, these data identify a novel role for p62 in trafficking nuclear proteins to nucleolar aggresomes under conditions of cell stress, thus maintaining nuclear proteostasis. They also provide invaluable information on the mechanisms that regulate the nuclear/nucleolar distribution of RelA that could be exploited for therapeutic purpose.SignificanceAberrant NF-κB activity drives many of the hallmarks of cancer and plays a key role in cancer progression. Nucleolar sequestration of NF-κB/RelA is one mechanism that switches off this activity and induces the death of cancer cells. Here we define a novel role for the autophagy receptor, SQSTM1/p62 in transport of nucleoplasmic NF-κB/RelA to nucleoli. Identification of this new trafficking mechanism opens up avenues for the development of a unique class of therapeutic agents that transport RelA and other cancer regulatory proteins to this organelle.

scholarly journals MBNL1 alternative splicing isoforms play opposing roles in cancer

2018 ◽  
Vol 1 (5) ◽  
pp. e201800157 ◽  
Author(s):  
Tommaso Tabaglio ◽  
Diana HP Low ◽  
Winnie Koon Lay Teo ◽  
Pierre Alexis Goy ◽  
Piotr Cywoniuk ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Cancer Cells ◽  
Cancer Progression ◽  
Gene Function ◽  
Dominant Negative ◽  
Individual Gene ◽  
Cancer Types ◽  
Splicing Isoforms ◽  
Isoform Switching ◽  
And Migration

The extent of and the oncogenic role played by alternative splicing (AS) in cancer are well documented. Nonetheless, only few studies have attempted to dissect individual gene function at an isoform level. Here, we focus on the AS of splicing factors during prostate cancer progression, as these factors are known to undergo extensive AS and have the potential to affect hundreds of downstream genes. We identified exon 7 (ex7) in the MBNL1 (Muscleblind-like 1) transcript as being the most differentially included exon in cancer, both in cell lines and in patients' samples. In contrast, MBNL1 overall expression was down-regulated, consistently with its described role as a tumor suppressor. This observation holds true in the majority of cancer types analyzed. We first identified components associated to the U2 splicing complex (SF3B1, SF3A1, and PHF5A) as required for efficient ex7 inclusion and we confirmed that this exon is fundamental for MBNL1 protein homodimerization. We next used splice-switching antisense oligonucleotides (AONs) or siRNAs to compare the effect of MBNL1 splicing isoform switching with knockdown. We report that whereas the absence of MBNL1 is tolerated in cancer cells, the expression of isoforms lacking ex7 (MBNL1 Δex7) induces DNA damage and inhibits cell viability and migration, acting as dominant negative proteins. Our data demonstrate the importance of studying gene function at the level of alternative spliced isoforms and support our conclusion that MBNL1 Δex7 proteins are antisurvival factors with a defined tumor suppressive role that cancer cells tend to down-regulate in favor of MBNL +ex7 isoforms.

scholarly journals Bridging angiogenesis and immune evasion in the hypoxic tumor microenvironment

2018 ◽  
Vol 315 (6) ◽  
pp. R1072-R1084 ◽  
Author(s):  
Luana Schito
Keyword(s):  
Tumor Microenvironment ◽  
Cancer Cells ◽  
Cancer Progression ◽  
Tumor Vasculature ◽  
Antiangiogenic Agents ◽  
Pathophysiological Mechanism ◽  
Feedforward Loop ◽  
Antiangiogenic Therapies ◽  
Solid Malignancies ◽  
Hypoxic Tumor

Hypoxia (low O2) is a ubiquitous microenvironmental factor promoting cancer progression, metastasis, and mortality, owing to the ability of cancer cells to co-opt physiological angiogenic responses. Notwithstanding, the pathophysiological induction of angiogenesis results in an abnormal tumor vasculature, further aggravating hypoxia in a feedforward loop that limits the efficacy of molecular targeted therapies. Recent studies suggest that, besides their canonical roles, angiogenic factors promote a panoply of immunosuppressive effects in the tumor microenvironment. Therefore, intratumoral hypoxia emerges as a hitherto unrecognized mechanism evolutionarily repurposing angiogenic molecules as (patho)physiological immunomodulators. On the other hand, antiangiogenic therapies could be aimed at impeding both tumor growth and immunotolerance toward cancer cells, a beneficial effect that can be countered if hypoxia signaling pathways are left unchecked, leading to therapeutic failure. This review summarizes evidence supporting the hypothesis that hypoxia acts as a common pathophysiological mechanism of resistance to immunotherapeutic and antiangiogenic agents while proposing potential strategies to curtail resistance and mortality in patients bearing solid malignancies.

scholarly journals Discovering and Characterizing of Survivin Dominant Negative Mutants With Stronger Pro-apoptotic Activity on Cancer Cells and CSCs

2021 ◽  
Vol 11 ◽  
Author(s):  
Wei Guo ◽  
Xingyuan Ma ◽  
Yunhui Fu ◽  
Chang Liu ◽  
Qiuli Liu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cancer Cells ◽  
A549 Cells ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Anti Cancer ◽  
Apoptotic Activity ◽  
Reversal Index ◽  
Negative Mutant

Survivin as a member of the inhibitor of apoptosis proteins (IAPs) family is undetectable in normal cells, but highly expressed in cancer cells and cancer stem cells (CSCs) which makes it an attractive target in cancer therapy. Survivin dominant negative mutants have been reported as competitive inhibitors of endogenous survivin protein in cancer cells. However, there is a lack of systematic comparative studies on which mutants have stronger effect on promoting apoptosis in cancer cells, which will hinder the development of novel anti-cancer drugs. Here, based on the previous study of survivin and its analysis of the relationship between structure and function, we designed and constructed a series of different amino acid mutants from survivin (TmSm34, TmSm48, TmSm84, TmSm34/48, TmSm34/84, and TmSm34/48/84) fused cell-permeable peptide TATm at the N-terminus, and a dominant negative mutant TmSm34/84 with stronger pro-apoptotic activity was selected and evaluated systematically in vitro. The double-site mutant of survivin (TmSm34/84) showed more robust pro-apoptotic activity against A549 cells than others, and could reverse the resistance of A549 CSCs to adriamycin (ADM) (reversal index up to 7.01) by decreasing the expression levels of survivin, P-gp, and Bcl-2 while increasing cleaved caspase-3 in CSCs. This study indicated the selected survivin dominant negative mutant TmSm34/84 is promising to be an excellent candidate for recombinant anti-cancer protein by promoting apoptosis of cancer cells and their stem cells and sensitizing chemotherapeutic drugs.

scholarly journals Signalling, cycling and desensitisation of gonadotrophin-releasing hormone receptors

2002 ◽  
Vol 173 (1) ◽  
pp. 1-11 ◽  
Author(s):  
CA McArdle ◽  
J Franklin ◽  
L Green ◽  
JN Hislop
Keyword(s):  
Cancer Cells ◽  
G Protein ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Type I ◽  
Pituitary Cells ◽  
Type Ii ◽  
Inhibition Of Proliferation ◽  
Gonadotrophin Secretion ◽  
Receptor Desensitisation

Sustained stimulation of G-protein-coupled receptors (GPCRs) typically causes receptor desensitisation, which is mediated by phosphorylation, often within the C-terminal tail of the receptor. The consequent binding of beta-arrestin not only prevents the receptor from activating its G protein (causing desensitisation), but can also target it for internalisation via clathrin-coated vesicles and can mediate signalling to proteins regulating endocytosis and mitogen-activated protein kinase (MAPK) cascades. GnRH acts via phospholipase C (PLC)-coupled GPCRs on pituitary gonadotrophs to stimulate a Ca(2+)-mediated increase in gonadotrophin secretion. The type I GnRH receptors (GnRH-Rs), found only in mammals, are unique in that they lack C-terminal tails and apparently do not undergo agonist-induced phosphorylation or bind beta-arrestin; they are therefore resistant to receptor desensitisation and internalise slowly. In contrast, the type II GnRH-Rs, found in numerous vertebrates, possess such tails and show rapid desensitisation and internalisation, with concomitant receptor phosphorylation (within the C-terminal tails) or binding of beta-arrestin, or both. The association with beta-arrestin may also be important for regulation of dynamin, a GTPase that controls separation of endosomes from the plasma membrane. Using recombinant adenovirus to express GnRH-Rs in Hela cells conditionally expressing a dominant negative mutant of dynamin (K44A), we have found that blockade of dynamin-dependent endocytosis inhibits internalisation of type II (xenopus) GnRH-Rs but not type I (human) GnRH-Rs. In these cells, blockade of dynamin-dependent internalisation also inhibited GnRH-R-mediated MAPK activation, but this effect was not receptor specific and therefore not dependent upon dynamin-regulated GnRH-R internalisation. Although type I GnRH-Rs do not desensitise, sustained activation of GnRH-Rs causes desensitisation of gonadotrophin secretion, and we have found that GnRH can cause down-regulation of inositol (1,4,5) trisphosphate receptors and desensitisation of Ca(2+) mobilisation in pituitary cells. The atypical resistance of the GnRH-R to desensitisation may underlie its atypical efficiency at provoking this downstream adaptive response. GnRH-Rs are also expressed in several extrapituitary sites, and these may mediate direct inhibition of proliferation of hormone-dependent cancer cells. Infection with type I GnRH-R-expressing adenovirus facilitated expression of high-affinity, PLC-coupled GnRH-R in mammary and prostate cancer cells, and these mediated pronounced antiproliferative effects of receptor agonists. No such effect was seen in cells transfected with a type II GnRH-R, implying that it is mediated most efficiently by a non-desensitising receptor. Thus it appears that the mammalian GnRH-Rs have undergone a period of rapidly accelerated molecular evolution that is of functional relevance to GnRH-Rs in pituitary and extrapituitary sites.

Inhibition of AP-1 suppresses cervical cancer cell proliferation and is associated with p21 expression

2011 ◽  
Vol 392 (5) ◽  
Author(s):  
Michelle F. Maritz ◽  
Pauline J. van der Watt ◽  
Nina Holderness ◽  
Michael J. Birrer ◽  
Virna D. Leaner
Keyword(s):  
Cell Cycle ◽  
Cervical Cancer ◽  
Cell Proliferation ◽  
Cancer Cells ◽  
Regulatory Protein ◽  
Fold Increase ◽  
Significant Inhibition ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Cervical Cancer Cells

AbstractAP-1, a transcription factor comprised primarily of Jun and Fos family proteins, regulates genes involved in proliferation, differentiation and oncogenesis. Previous studies demonstrated that elevated expression of Jun and Fos family member proteins is associated with numerous human cancers and in cancer-relevant biological processes. In this study we used a dominant-negative mutant of c-Jun, Tam67, which interferes with the functional activity of all AP-1 complexes, to investigate the requirement of AP-1 in the proliferation and cell cycle progression of cervical cancer cells. Transient and stable expression of Tam67 in CaSki cervical cancer cells resulted in decreased AP-1 activity that correlated with a significant inhibition of cell proliferation and anchorage-independent colony formation. Inhibiting AP-1 activity resulted in a two-fold increase in cells located in the G2/M phase of the cell cycle and an accompanying increase in the expression of the cell cycle regulatory protein, p21. The increase in p21 was associated with a decrease in HPV E6 expression and an increase in p53. Importantly, blocking the induction of p21 in CaSki-Tam67-expressing cells accelerated their proliferation rate to that of CaSki, implicating p21 as a key player in the growth arrest induced by Tam67. Our results suggest a role for AP-1 in the proliferation, G2/M progression and inhibition of p21 expression in cervical cancer.

scholarly journals E5 transforming proteins of papillomaviruses do not disturb the activity of the vacuolar H+-ATPase

2001 ◽  
Vol 82 (10) ◽  
pp. 2353-2362 ◽  
Author(s):  
Andrew D. M. Ashby ◽  
Liam Meagher ◽  
M. Saveria Campo ◽  
Malcolm E. Finbow
Keyword(s):  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Type I ◽  
Bovine Papillomavirus ◽  
Subunit C ◽  
T Lymphotropic Virus ◽  
Transforming Activity ◽  
Transforming Proteins ◽  
Related Proteins ◽  
Vacuolar Membranes

Papillomaviruses contain a gene, E5, that encodes a short hydrophobic polypeptide that has transforming activity. E5 proteins bind to the 16 kDa subunit c (proteolipid) of the eukaryotic vacuolar H+-ATPase (V-ATPase) and this binding is thought to disturb the V-ATPase and to be part of transformation. This link has been examined in the yeast Saccharomyces cerevisiae. The E5 proteins from human papillomavirus (HPV) type 16, bovine papillomavirus (BPV) type 1, BPV-4 E5 and various mutants of E5 and the p12′ polypeptide from human T-lymphotropic virus (HTLV) type I all bound to the S. cerevisiae subunit c (Vma3p) and could be found in vacuolar membranes. However, none affected the activity of the V-ATPase. In contrast, a dominant-negative mutant of Vma3p (E137G) inactivated the enzyme and gave the characteristic VMA phenotype. A hybrid V-ATPase containing a subunit c from Norway lobster also showed no disruption. Sedimentation showed that HPV-16 E5 was not part of the active V-ATPase. It is concluded that the binding of E5 and E5-related proteins to subunit c does not affect V-ATPase activity or function and it is proposed that the binding may be due to a chaperone function of subunit c.

scholarly journals The GTPase KRAS suppresses the p53 tumor suppressor by activating the NRF2-regulated antioxidant defense system in cancer cells

2020 ◽  
Vol 295 (10) ◽  
pp. 3055-3063 ◽  
Author(s):  
Hua Yang ◽  
Shengyan Xiang ◽  
Aslamuzzaman Kazi ◽  
Said M. Sebti
Keyword(s):  
Cancer Cells ◽  
Antioxidant Defense ◽  
Antioxidant Defense System ◽  
Dominant Negative ◽  
Human Lung Cancer ◽  
Dominant Negative Mutant ◽  
Related Factor ◽  
Dependent Manner ◽  
Defense System ◽  
Iκb Kinase

In human cancer cells that harbor mutant KRAS and WT p53 (p53), KRAS contributes to the maintenance of low p53 levels. Moreover, KRAS depletion stabilizes and reactivates p53 and thereby inhibits malignant transformation. However, the mechanism by which KRAS regulates p53 is largely unknown. Recently, we showed that KRAS depletion leads to p53 Ser-15 phosphorylation (P-p53) and increases the levels of p53 and its target p21/WT p53-activated fragment 1 (WAF1)/CIP1. Here, using several human lung cancer cell lines, siRNA-mediated gene silencing, immunoblotting, quantitative RT-PCR, promoter–reporter assays, and reactive oxygen species (ROS) assays, we demonstrate that KRAS maintains low p53 levels by activating the NRF2 (NFE2-related factor 2)–regulated antioxidant defense system. We found that KRAS depletion led to down-regulation of NRF2 and its targets NQO1 (NAD(P)H quinone dehydrogenase 1) and SLC7A11 (solute carrier family 7 member 11), decreased the GSH/GSSG ratio, and increased ROS levels. We noted that the increase in ROS is required for increased P-p53, p53, and p21Waf1/cip1 levels following KRAS depletion. Downstream of KRAS, depletion of RalB (RAS-like proto-oncogene B) and IκB kinase–related TANK-binding kinase 1 (TBK1) activated p53 in a ROS- and NRF2-dependent manner. Consistent with this, the IκB kinase inhibitor BAY11-7085 and dominant-negative mutant IκBαM inhibited NF-κB activity and increased P-p53, p53, and p21Waf1/cip1 levels in a ROS-dependent manner. In conclusion, our findings uncover an important role for the NRF2-regulated antioxidant system in KRAS-mediated p53 suppression.

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