Regulation of HDM2 E3-ubiquitin ligase in esophageal adenocarcinoma cells by AURKA.

35 Background: Esophageal adenocarcinomas (EAC) exhibit intrinsic resistance against chemotherapy. AURKA regulates cell cycle progression and its overexpression is associated with oncogenic transformation. We have recently reported that AURKA is significantly overexpressed in about 70% of human EAC tissue samples and EAC cell lines. We have previously shown that AURKA inhibits p53- and p73-mediated apoptotic pathways in GI adenocarcinomas. HDM2 is an E3-ubiquitin ligase which is closely involved in regulating p53 and p73 protein stability and activity. In this study we demonstrate that AURKA directly interacts with HDM2 and regulates HDM2 protein expression and phosphorylation in both FLO-1 and OE33 EAC cells. Methods and Results: Western blot analyses were done following AURKA overexpression with adenovirus, knockdown with si-RNA or inhibition with MLN 8237 (0.5µM) in FLO-1 and OE33 EAC cell lines. The data indicated that overexpression of AURKA induced both total and phospho-HDM2-(Ser166) protein levels. Knockdown or inhibition of AURKA significantly decreased expression of both total and phospho-HDM2-(Ser166) protein levels in FLO-1 and OE33 EAC cells. Additionally, following adenovirus mediated overexpression of AURKA, co-immunoprecipitaion (Co-IP) was done for AURKA and HDM2 in FLO-1 and OE33 EAC cells. The two-way Co-IP data indicated the presence of HDM2 in a complex associated with AURKA and vice-versa. The data from in vitro protein kinase assay indicated that recombinant AURKA directly phosphorylates recombinant HDM2 at Ser166 site. To confirm direct interaction between recombinant AURKA and HDM2 proteins we performed IP following the in vitro kinase assay. The in vitro kinase IP data indicates that kinase intact recombinant AURKA directly interacts and phosphorylates recombinant HDM2 protein. Conclusions: Our data indicate that AURKA regulates HDM2 expression and phosphorylation in both FLO-1 and OE33 EAC cells. Additionally, we also report for the first time that AURKA directly interacts with HDM2 and phosphorylates it at Ser166 site. Therefore, our study suggests that AURKA-mediated regulation of HDM2 could be the major underlying mechanism for induction of apoptosis in p53-negative EAC.

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An E3 Ubiquitin Ligase RNF139 Serves as a Tumor-Suppressor in Glioma

Journal of Molecular Neuroscience ◽

10.1007/s12031-021-01860-4 ◽

2021 ◽

Author(s):

Xiaofeng Chen ◽

Weiping Kuang ◽

Yong Zhu ◽

Bin Zhou ◽

Xiaosong Li ◽

...

Keyword(s):

Tumor Suppressor ◽

Cell Lines ◽

Ubiquitin Ligase ◽

Glioma Cell ◽

Protein Modification ◽

E3 Ubiquitin Ligase ◽

Ring Finger ◽

Glioma Cells ◽

Migration And Invasion ◽

Tissue Samples

AbstractGlioma is highly lethal because of its high malignancy. Ubiquitination, a type of ubiquitin-dependent protein modification, has been reported to play an oncogenic or tumor-suppressive role in glioma development, depending on the targets. Ring finger protein 139 (RNF139) is a membrane-bound E3 ubiquitin ligase serving as a tumor suppressor by ubiquitylation-dependently suppressing cell growth. Herein, we firstly confirmed the abnormal downregulation of RNF139 in glioma tissues and cell lines. In glioma cells, ectopic RNF139 overexpression could inhibit, whereas RNF139 knockdown could aggravate the aggressive behaviors of glioma cells, including hyperproliferation, migration, and invasion. Moreover, in two glioma cell lines, RNF139 overexpression inhibited, whereas RNF139 knockdown enhanced the phosphorylation of phosphatidylinositol 3-kinase (PI3K) and AKT serine/threonine kinase 1 (AKT). In a word, we demonstrate the aberration in RNF139 expression in glioma tissue samples and cell lines. RNF139 serves as a tumor-suppressor in glioma by inhibiting glioma cell proliferation, migration, and invasion and promoting glioma cell apoptosis through regulating PI3K/AKT signaling.

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CBL mutations drive PI3K/AKT signaling via increased interaction with LYN and PIK3R1

Blood ◽

10.1182/blood.2020006528 ◽

2021 ◽

Author(s):

Roger Belizaire ◽

Sebastian Hassan John Koochaki ◽

Namrata D. Udeshi ◽

Alexis Vedder ◽

Lei Sun ◽

...

Keyword(s):

Cell Lines ◽

Ubiquitin Ligase ◽

Therapeutic Potential ◽

Mutant Cell ◽

E3 Ubiquitin Ligase ◽

Akt Signaling ◽

Allelic Series ◽

Genetic Ablation

CBL encodes an E3 ubiquitin ligase and signaling adaptor that regulates receptor and non-receptor tyrosine kinases. Recurrent CBL mutations occur in myeloid neoplasms, including 10-20% of chronic myelomonocytic leukemia (CMML) cases, and selectively disrupt the protein's E3 ubiquitin ligase activity. CBL mutations have been associated with poor prognosis, but the oncogenic mechanisms and therapeutic implications of CBL mutations remain incompletely understood. We combined functional assays and global mass spectrometry to define the phosphoproteome, CBL interactome, and mechanism of signaling activation in a panel of cell lines expressing an allelic series of CBL mutations. Our analyses revealed that increased LYN activation and interaction with mutant CBL are key drivers of enhanced CBL phosphorylation, PIK3R1 recruitment, and downstream PI3K/AKT signaling in CBL-mutant cells. Signaling adaptor domains of CBL, including the tyrosine-kinase binding domain, proline-rich region, and C-terminal phosphotyrosine sites, were all required for the oncogenic function of CBL mutants. Genetic ablation or dasatinib-mediated inhibition of LYN reduced CBL phosphorylation, CBL-PIK3R1 interaction, and PI3K/AKT signaling. Furthermore, we demonstrated in vitro and in vivo antiproliferative efficacy of dasatinib in CBL-mutant cell lines and primary CMML. Overall, these mechanistic insights into the molecular function of CBL mutations provide rationale to explore the therapeutic potential of LYN inhibition in CBL-mutant myeloid malignancies.

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Lenalidomide Induces Ubiquitination and Degradation of CSNK1A1 in MDS with Del(5q)

Blood ◽

10.1182/blood.v124.21.4.4 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4-4 ◽

Cited By ~ 6

Author(s):

Emma C. Fink ◽

Jan Krönke ◽

Slater N. Hurst ◽

Namrata D. Udeshi ◽

Tanya Svinkina ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Conditional Knockout ◽

Therapeutic Window ◽

Mrna Levels ◽

Dependent Manner ◽

Wild Type ◽

Protein Levels

Abstract The immunomodulatory (IMiD) drug lenalidomide is a highly effective treatment for multiple myeloma and myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)). Recently, we and others demonstrated that lenalidomide activates the CRBN-CRL4 E3 ubiquitin ligase to ubiquitinate IKZF1 and IKZF3. Degradation of these lymphoid transcription factors explains lenalidomide’s growth inhibition of multiple myeloma cells and increased IL-2 release from T cells. However, it is unlikely that degradation of IKZF1 and IKZF3 accounts for lenalidomide’s activity in MDS with del(5q). Instead, we hypothesized that ubiquitination of a distinct CRBN substrate in myeloid cells explains the efficacy of lenalidomide in del(5q) MDS. Applying quantitative proteomics in the myeloid cell line KG-1, we identified a novel target, casein kinase 1A1 (CSNK1A1), that had increased ubiquitination and decreased protein abundance following lenalidomide treatment. CSNK1A1 is encoded in the del(5q) commonly deleted region and is thus a potential lenalidomide target in del(5q) MDS. Previous studies have demonstrated that Csnk1a1 is a therapeutic target in a murine model of acute myeloid leukemia. We validated that lenalidomide treatment decreased CSNK1A1 protein levels in multiple human cell lines in a dose-dependent manner without altering CSNK1A1 mRNA levels. Moreover, lenalidomide treatment increased ubiquitination of CSNK1A1 in cell lines. The decrease in CSNK1A1 protein levels in response to lenalidomide was abrogated by treatment with the proteasome inhibitor MG132 and by Cullin-RING ubiquitin ligase inhibition with MLN4924. CSNK1A1 co-immunoprecipitated with CRBN in the presence of lenalidomide, demonstrating direct interaction of CSNK1A1 with the substrate adaptor for the ubiquitin ligase. Homozygous genetic inactivation of the CRBN gene by CRISPR/Cas9 genome editing in 293T cells eliminated lenalidomide-induced degradation of CSNK1A1. In aggregate, these experiments demonstrate that CSNK1A1 is a CRBN-CRL4 substrate that is ubiquitinated and degraded in the presence of lenalidomide. We next explored how degradation of CSNK1A1 might explain the specificity of lenalidomide for cells with del(5q). ShRNA-mediated knockdown of CSNK1A1 sensitized primary human CD34+ cells to lenalidomide treatment, indicating that haploinsufficiency for CSNK1A1 might increase lenalidomide sensitivity in del(5q) hematopoietic cells. We sought to further validate this finding in a genetically defined Csnk1a1 conditional knockout mouse model. While murine cells are resistant to the effects of IMiDs, murine Ba/F3 cells overexpressing human CRBN (hCRBN), but not murine CRBN, degraded CSNK1A1 in response to lenalidomide. To examine the effect of Csnk1a1 haploinsufficiency on lenalidomide sensitivity, we isolated hematopoietic stem and progenitor cells from Csnk1a1+/- and Csnk1a1+/+ mice and transduced them with a retroviral vector expressing hCRBN. When treated with lenalidmide, Csnk1a1+/- cells expressing hCRBN were depleted over time relative to wild-type controls. The enhanced sensitivity of Csnk1a1+/- cells to lenalidomide was associated with induction of p21 and was rescued by heterozygous deletion of p53, demonstrating a critical downstream role for p53 consistent with clinical observations that TP53 mutations confer lenalidomide resistance. In aggregate, these studies demonstrate that lenalidomide induces the ubiquitination and consequent degradation of CSNK1A1 by the CRBN-CRL4 E3 ubiquitin ligase. del(5q) cells have only one copy of CSNK1A1, so they are selectively depleted over wild-type cells, explaining lenalidomide’s clinical efficacy in del(5q) MDS. Although the idea that heterozygous deletions could be cancer vulnerabilities was first proposed 20 years ago, lenalidomide provides the first example of an FDA-approved and clinically effective drug that derives its therapeutic window from specifically targeting a haploinsufficient gene. Disclosures Ebert: Celgene: Research Funding; Genoptix: Consultancy.

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Design, Synthesis, Molecular Docking, and Anticancer Evaluation of Pyrazole Linked Pyrazoline Derivatives with Carbothioamide Tail as EGFR Kinase Inhibitors

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620666200727093613 ◽

2020 ◽

Vol 21 (1) ◽

pp. 42-60

Author(s):

Farah Nawaz ◽

Ozair Alam ◽

Ahmad Perwez ◽

Moshahid A. Rizvi ◽

Mohd. Javed Naim ◽

...

Keyword(s):

Molecular Docking ◽

Cell Line ◽

Cell Lines ◽

Cancer Cell ◽

Cancer Cell Line ◽

Cancer Cell Lines ◽

Kinase Assay ◽

Cervix Uteri ◽

Egfr Kinase

Background: The Epidermal Growth Factor Receptor (known as EGFR) induces cell differentiation and proliferation upon activation through the binding of its ligands. Since EGFR is thought to be involved in the development of cancer, the identification of new target inhibitors is the most viable approach, which recently gained momentum as a potential anticancer therapy. Objective: To assess various pyrazole linked pyrazoline derivatives with carbothioamide for EGFR kinase inhibitory as well as anti-proliferative activity against human cancer cell lines viz. A549 (non-small cell lung tumor), MCF-7 (breast cancer cell line), SiHa (cancerous tissues of the cervix uteri), and HCT-116 (colon cancer cell line). Methods: In vitro EGFR kinase assay, in vitro MTT assay, Lactate dehydrogenase release, nuclear staining (DAPI), and flow cytometry cell analysis. Results: Compounds 6h and 6j inhibited EGFR kinase at concentrations of 1.66μM and 1.9μM, respectively. Furthermore, compounds 6h and 6j showed the most potent anti-proliferative results against the A549 KRAS mutation cell line (IC50 = 9.3 & 10.2μM). Through DAPI staining and phase contrast microscopy, it was established that compounds 6h and 6j also induced apoptotic activity in A549 cells. This activity was further confirmed by FACS using Annexin-V-FITC and Propidium Iodide (PI) labeling. Molecular docking studies performed on 6h and 6j suggested that the compounds can bind to the hinge region of ATP binding site of EGFR tyrosine kinase in a similar pose as that of the standard drug gefitinib. Conclusion: The potential anticancer activity of compounds 6h and 6j was confirmed and need further exploration in cancer cell lines of different tissue origin and signaling pathways, as well as in animal models of cancer development.

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TRIM27 interacts with Iκbα to promote the growth of human renal cancer cells through regulating the NF-κB pathway

BMC Cancer ◽

10.1186/s12885-021-08562-5 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Chengwu Xiao ◽

Wei Zhang ◽

Meimian Hua ◽

Huan Chen ◽

Bin Yang ◽

...

Keyword(s):

Cell Lines ◽

Prognostic Indicator ◽

The Cancer Genome Atlas ◽

Mrna Levels ◽

Loss Of Function ◽

Protein Levels ◽

Kaplan Meier ◽

Cancer Genome Atlas

Abstract Background The tripartite motif (TRIM) family proteins exhibit oncogenic roles in various cancers. The roles of TRIM27, a member of the TRIM super family, in renal cell carcinoma (RCC) remained unexplored. In the current study, we aimed to investigate the clinical impact and roles of TRIM27 in the development of RCC. Methods The mRNA levels of TRIM27 and Kaplan–Meier survival of RCC were analyzed from The Cancer Genome Atlas database. Real-time PCR and Western blotting were used to measure the mRNA and protein levels of TRIM27 both in vivo and in vitro. siRNA and TRIM27 were exogenously overexpressed in RCC cell lines to manipulate TRIM27 expression. Results We discovered that TRIM27 was elevated in RCC patients, and the expression of TRIM27 was closely correlated with poor prognosis. The loss of function and gain of function results illustrated that TRIM27 promotes cell proliferation and inhibits apoptosis in RCC cell lines. Furthermore, TRIM27 expression was positively associated with NF-κB expression in patients with RCC. Blocking the activity of NF-κB attenuated the TRIM27-mediated enhancement of proliferation and inhibition of apoptosis. TRIM27 directly interacted with Iκbα, an inhibitor of NF-κB, to promote its ubiquitination, and the inhibitory effects of TRIM27 on Iκbα led to NF-κB activation. Conclusions Our results suggest that TRIM27 exhibits an oncogenic role in RCC by regulating NF-κB signaling. TRIM27 serves as a specific prognostic indicator for RCC, and strategies targeting the suppression of TRIM27 function may shed light on future therapeutic approaches.

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E3 Ubiquitin Ligase SPL2 Is a Lanthanide-Binding Protein

International Journal of Molecular Sciences ◽

10.3390/ijms22115712 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5712

Author(s):

Michał Tracz ◽

Ireneusz Górniak ◽

Andrzej Szczepaniak ◽

Wojciech Białek

Keyword(s):

Ubiquitin Ligase ◽

Conformational Changes ◽

Protein Interactions ◽

E3 Ubiquitin Ligase ◽

Lanthanide Ions ◽

E3 Ligases ◽

Fluorescence Measurements ◽

Partial Unfolding

The SPL2 protein is an E3 ubiquitin ligase of unknown function. It is one of only three types of E3 ligases found in the outer membrane of plant chloroplasts. In this study, we show that the cytosolic fragment of SPL2 binds lanthanide ions, as evidenced by fluorescence measurements and circular dichroism spectroscopy. We also report that SPL2 undergoes conformational changes upon binding of both Ca2+ and La3+, as evidenced by its partial unfolding. However, these structural rearrangements do not interfere with SPL2 enzymatic activity, as the protein retains its ability to auto-ubiquitinate in vitro. The possible applications of lanthanide-based probes to identify protein interactions in vivo are also discussed. Taken together, the results of this study reveal that the SPL2 protein contains a lanthanide-binding site, showing for the first time that at least some E3 ubiquitin ligases are also capable of binding lanthanide ions.

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HERV-K Gag RNA and Protein Levels Are Elevated in Malignant Regions of the Prostate in Males with Prostate Cancer

Viruses ◽

10.3390/v13030449 ◽

2021 ◽

Vol 13 (3) ◽

pp. 449

Author(s):

Simin D. Rezaei ◽

Joshua A. Hayward ◽

Sam Norden ◽

John Pedersen ◽

John Mills ◽

...

Keyword(s):

Prostate Cancer ◽

Cell Lines ◽

Endogenous Retrovirus ◽

Human Endogenous Retrovirus ◽

Normal Prostate ◽

Tissue Samples ◽

Gag Protein ◽

Protein Levels ◽

Prostate Epithelial Cells ◽

Prostate Cancers

Heightened expression of human endogenous retrovirus (HERV) sequences has been associated with a range of malignancies, including prostate cancer, suggesting that they may serve as useful diagnostic or prognostic cancer biomarkers. We analysed the expression of HERV-K (Gag and Env/Np9 regions), HERV-E 4.1 (Pol and Env regions), HERV-H (Pol) and HERV-W (Gag) sequences in prostate cancer cells lines and normal prostate epithelial cells using qRT-PCR. HERV expression was also analysed in matched malignant and benign prostate tissue samples from men with prostate cancer (n = 27, median age 65.2 years (range 47–70)) and compared to prostate cancer-free male controls (n = 11). Prostate cancer epithelial cell lines exhibited a signature of HERV RNA overexpression, with all HERVs analysed, except HERV-E Pol, showing heightened expression in at least two, but more commonly all, cell lines analysed. Analysis of primary prostate material indicated increased expression of HERV-E Pol but decreased expression of HERV-E Env in both malignant and benign regions of the prostate in men with prostate cancer as compared to those without. Expression of HERV-K Gag was significantly higher in malignant regions of the prostate in men with prostate cancer as compared to matched benign regions and prostate cancer-free men (p < 0.001 for both), with 85.2% of prostate cancers donors showing malignancy-associated upregulation of HERV-K Gag RNA. HERV-K Gag protein was detected in 12/18 (66.7%) malignant tissues using immunohistochemistry, but only 1/18 (5.6%) benign tissue sections. Heightened expression of HERV-K Gag RNA and protein appears to be a sensitive and specific biomarker of prostate malignancy in this cohort of men with prostate carcinoma, supporting its potential utility as a non-invasive, adjunct clinical biomarker.

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Inhibition of the Myocardin-Related Transcription Factor Pathway Increases Efficacy of Trametinib in NRAS-Mutant Melanoma Cell Lines

Cancers ◽

10.3390/cancers13092012 ◽

2021 ◽

Vol 13 (9) ◽

pp. 2012

Author(s):

Kathryn M. Appleton ◽

Charuta C. Palsuledesai ◽

Sean A. Misek ◽

Maja Blake ◽

Joseph Zagorski ◽

...

Keyword(s):

Cell Lines ◽

Melanoma Cell ◽

Therapeutic Potential ◽

Mapk Pathway ◽

Mek Inhibitor ◽

Intrinsic Resistance ◽

Primary Resistance ◽

Induced Apoptosis ◽

Melanoma Cell Lines

The Ras/MEK/ERK pathway has been the primary focus of targeted therapies in melanoma; it is aberrantly activated in almost 80% of human cutaneous melanomas (≈50% BRAFV600 mutations and ≈30% NRAS mutations). While drugs targeting the MAPK pathway have yielded success in BRAFV600 mutant melanoma patients, such therapies have been ineffective in patients with NRAS mutant melanomas in part due to their cytostatic effects and primary resistance. Here, we demonstrate that increased Rho/MRTF-pathway activation correlates with high intrinsic resistance to the MEK inhibitor, trametinib, in a panel of NRAS mutant melanoma cell lines. A combination of trametinib with the Rho/MRTF-pathway inhibitor, CCG-222740, synergistically reduced cell viability in NRAS mutant melanoma cell lines in vitro. Furthermore, the combination of CCG-222740 with trametinib induced apoptosis and reduced clonogenicity in SK-Mel-147 cells, which are highly resistant to trametinib. These findings suggest a role of the Rho/MRTF-pathway in intrinsic trametinib resistance in a subset of NRAS mutant melanoma cell lines and highlight the therapeutic potential of concurrently targeting the Rho/MRTF-pathway and MEK in NRAS mutant melanomas.

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Bortezomib blocks Bax degradation in malignant B cells during treatment with TRAIL

Blood ◽

10.1182/blood-2007-08-110445 ◽

2008 ◽

Vol 111 (5) ◽

pp. 2797-2805 ◽

Cited By ~ 57

Author(s):

Feng-Ting Liu ◽

Samir G. Agrawal ◽

John G. Gribben ◽

Hongtao Ye ◽

Ming-Qing Du ◽

...

Keyword(s):

B Cells ◽

Cell Lines ◽

Proteasome Inhibitor ◽

Resistant Cell ◽

Protein Levels ◽

Bax Protein ◽

Degradation Activity ◽

Potent Drug ◽

Induced Apoptosis

Proapoptotic Bcl-2 family member Bax is a crucial protein in the induction of apoptosis, and its activation is required for this process. Here we report that Bax is a short-lived protein in malignant B cells and Bax protein levels decreased rapidly when protein synthesis was blocked. Malignant B cells were relatively resistant to tumor necrosis factor–related apoptosis inducing ligand (TRAIL)–induced apoptosis, and this correlated with low basal Bax protein levels. Furthermore, during treatment with TRAIL, the resistant cell lines showed prominent Bax degradation activity. This degradation activity was localized to mitochondrial Bax and could be prevented by truncated Bid, a BH3-only protein; in contrast, cytosolic Bax was relatively stable. The proteasome inhibitor bortezomib is a potent drug in inducing apoptosis in vitro in malignant B-cell lines and primary chronic lymphocytic leukemic (CLL) cells. In CLL cells, bortezomib induced Bax accumulation, translocation to mitochondria, conformational change, and oligomerization. Accumulation and stabilization of Bax protein by bortezomib-sensitized malignant B cells to TRAIL-induced apoptosis. This study reveals that Bax instability confers resistance to TRAIL, which can be reversed by Bax stabilization with a proteasome inhibitor.

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