Publications by authors named "Shyam Kumar Gudey"

10 Publications

  • Page 1 of 1

TRAF6 function as a novel co-regulator of Wnt3a target genes in prostate cancer.

EBioMedicine 2019 Jul 28;45:192-207. Epub 2019 Jun 28.

Medical Biosciences, Umeå University, Umeå, Sweden. Electronic address:

Background: Tumour necrosis factor receptor associated factor 6 (TRAF6) promotes inflammation in response to various cytokines. Aberrant Wnt3a signals promotes cancer progression through accumulation of β-Catenin. Here we investigated a potential role for TRAF6 in Wnt signaling.

Methods: TRAF6 expression was silenced by siRNA in human prostate cancer (PC3U) and human colorectal SW480 cells and by CRISPR/Cas9 in zebrafish. Several biochemical methods and analyses of mutant phenotype in zebrafish were used to analyse the function of TRAF6 in Wnt signaling.

Findings: Wnt3a-treatment promoted binding of TRAF6 to the Wnt co-receptors LRP5/LRP6 in PC3U and LNCaP cells in vitro. TRAF6 positively regulated mRNA expression of β-Catenin and subsequent activation of Wnt target genes in PC3U cells. Wnt3a-induced invasion of PC3U and SW480 cells were significantly reduced when TRAF6 was silenced by siRNA. Database analysis revealed a correlation between TRAF6 mRNA and Wnt target genes in patients with prostate cancer, and high expression of LRP5, TRAF6 and c-Myc correlated with poor prognosis. By using CRISPR/Cas9 to silence TRAF6 in zebrafish, we confirm TRAF6 as a key molecule in Wnt3a signaling for expression of Wnt target genes.

Interpretation: We identify TRAF6 as an important component in Wnt3a signaling to promote activation of Wnt target genes, a finding important for understanding mechanisms driving prostate cancer progression. FUND: KAW 2012.0090, CAN 2017/544, Swedish Medical Research Council (2016-02513), Prostatacancerförbundet, Konung Gustaf V:s Frimurarestiftelse and Cancerforskningsfonden Norrland. The funders did not play a role in manuscript design, data collection, data analysis, interpretation nor writing of the manuscript.
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http://dx.doi.org/10.1016/j.ebiom.2019.06.046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6642315PMC
July 2019

Pro-invasive properties of Snail1 are regulated by sumoylation in response to TGFβ stimulation in cancer.

Oncotarget 2017 Nov 9;8(58):97703-97726. Epub 2017 Aug 9.

Department of Medical Biosciences, Umeå University, Umeå, Sweden.

Transforming growth factor β (TGFβ) is a key regulator of epithelial-to-mesenchymal transition (EMT) during embryogenesis and in tumors. The effect of TGFβ, on ΕΜΤ, is conveyed by induction of the pro-invasive transcription factor Snail1. In this study, we report that TGFβ stimulates Snail1 sumoylation in aggressive prostate, breast and lung cancer cells. Sumoylation of Snail1 lysine residue 234 confers its transcriptional activity, inducing the expression of classical EMT genes, as well as TGFβ receptor I (TβRI) and the transcriptional repressor Hes1. Mutation of Snail1 lysine residue 234 to arginine (K234R) abolished sumoylation of Snail1, as well as its migratory and invasive properties in human prostate cancer cells. An increased immunohistochemical expression of Snail1, Sumo1, TβRI, Hes1, and c-Jun was observed in aggressive prostate cancer tissues, consistent with their functional roles in tumorigenesis.
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http://dx.doi.org/10.18632/oncotarget.20097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716685PMC
November 2017

The Role of Ubiquitination to Determine Non-Smad Signaling Responses.

Methods Mol Biol 2016 ;1344:355-63

Department of Medical Biosciences, Umeå University, Pathology Building 6M, 2nd Floor, Umeå, 901 85, Sweden.

Ubiquitination is a posttranslational modification of proteins which acts as a key regulator of their function as well as fate. We have recently reported transforming growth factor β (TGFβ)-induced activation of non-Smad signaling responses through a specific Lys63-linked polyubiquitination of TGFβ type I receptor and TGFβ-associated kinase 1 (TAK1) that are utilized to specify cellular responses in cancer cells. This chapter gives a brief introduction of the biological importance of ubiquitination of proteins, the methods we have used for detecting new partners in the TGFβ signaling pathway and for performing ubiquitination assays.
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http://dx.doi.org/10.1007/978-1-4939-2966-5_23DOI Listing
May 2016

Targeting glucosylceramide synthase induction of cell surface globotriaosylceramide (Gb3) in acquired cisplatin-resistance of lung cancer and malignant pleural mesothelioma cells.

Exp Cell Res 2015 Aug 22;336(1):23-32. Epub 2015 May 22.

Department of Medical Biosciences, Umeå University, S-901 85 Umea, Sweden.

Background: Acquired resistance to cisplatin treatment is a caveat when treating patients with non-small cell lung cancer (NSCLC) and malignant pleural mesothelioma (MPM). Ceramide increases in response to chemotherapy, leading to proliferation arrest and apoptosis. However, a tumour stress activation of glucosylceramide synthase (GCS) follows to eliminate ceramide by formation of glycosphingolipids (GSLs) such as globotriaosylceramide (Gb3), the functional receptor of verotoxin-1. Ceramide elimination enhances cell proliferation and apoptosis blockade, thus stimulating tumor progression. GSLs transactivate multidrug resistance 1/P-glycoprotein (MDR1) and multidrug resistance-associated protein 1 (MRP1) expression which further prevents ceramide accumulation and stimulates drug efflux. We investigated the expression of Gb3, MDR1 and MRP1 in NSCLC and MPM cells with acquired cisplatin resistance, and if GCS activity or MDR1 pump inhibitors would reduce their expression and reverse cisplatin-resistance.

Methods: Cell surface expression of Gb3, MDR1 and MRP1 and intracellular expression of MDR1 and MRP1 was analyzed by flow cytometry and confocal microscopy on P31 MPM and H1299 NSCLC cells and subline cells with acquired cisplatin resistance. The effect of GCS inhibitor PPMP and MDR1 pump inhibitor cyclosporin A for 72h on expression and cisplatin cytotoxicity was tested.

Results: The cisplatin-resistant cells expressed increased cell surface Gb3. Cell surface Gb3 expression of resistant cells was annihilated by PPMP whereas cyclosporin A decreased Gb3 and MDR1 expression in H1299 cells. No decrease of MDR1 by PPMP was noted in using flow cytometry, whereas a decrease of MDR1 in H1299 and H1299res was indicated with confocal microscopy. No certain co-localization of Gb3 and MDR1 was noted. PPMP, but not cyclosporin A, potentiated cisplatin cytotoxicity in all cells.

Conclusions: Cell surface Gb3 expression is a likely tumour biomarker for acquired cisplatin resistance of NSCLC and MPM cells. Tumour cell resistance to MDR1 inhibitors of cell surface MDR1 and Gb3 could explain the aggressiveness of NSCLC and MPM. Therapy with GCS activity inhibitors or toxin targeting of the Gb3 receptor may substantially reduce acquired cisplatin drug resistance of NSCLC and MPM cells.
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http://dx.doi.org/10.1016/j.yexcr.2015.05.012DOI Listing
August 2015

TRAF6 promotes TGFβ-induced invasion and cell-cycle regulation via Lys63-linked polyubiquitination of Lys178 in TGFβ type I receptor.

Cell Cycle 2015 ;14(4):554-65

a Medical Biosciences ; Umeå University ; Umeå , Sweden.

Transforming growth factor β (TGFβ) can act either as a tumor promoter or a tumor suppressor in a context-dependent manner. High levels of TGFβ are found in prostate cancer tissues and correlate with poor patient prognosis. We recently identified a novel TGFβ-regulated signaling cascade in which TGFβ type I receptor (TβRI) is activated by the E3 ligase TNF-receptor-associated factor 6 (TRAF6) via the Lys63-linked polyubiquitination of TβRI. TRAF6 also contributes to activation of TNF-α-converting enzyme and presenilin-1, resulting in the proteolytic cleavage of TβRI and releasing the intracellular domain of TβRI, which is translocated to the nucleus to promote tumor invasiveness. In this report, we provide evidence that Lys178 of TβRI is polyubiquitinated by TRAF6. Moreover, our data suggest that TRAF6-mediated Lys63-linked ubiquitination of the TβRI intracellular domain is a prerequisite for TGFβ regulation of mRNA for cyclin D1 (CCND1), expression, as well as for the regulation of other genes controlling the cell cycle, differentiation, and invasiveness of prostate cancer cells.
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http://dx.doi.org/10.4161/15384101.2014.990302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4347693PMC
December 2015

TGFβ-induced invasion of prostate cancer cells is promoted by c-Jun-dependent transcriptional activation of Snail1.

Cell Cycle 2014 ;13(15):2400-14

a Ludwig Institute for Cancer Research; Science for Life Laboratory; Uppsala University; Uppsala, Sweden.

High levels of transforming growth factor-β (TGFβ) correlate with poor prognosis for patients with prostate cancer and other cancers. TGFβ is a multifunctional cytokine and crucial regulator of cell fate, such as epithelial to mesenchymal transition (EMT), which is implicated in cancer invasion and progression. TGFβ conveys its signals upon binding to type I and type II serine/threonine kinase receptors (TβRI/II); phosphorylation of Smad2 and Smad3 promotes their association with Smad4, which regulates expression of targets genes, such as Smad7, p21, and c-Jun. TGFβ also activates the ubiquitin ligase tumor necrosis factor receptor-associated factor 6 (TRAF6), which associates with TβRI and activates the p38 mitogen-activated protein kinase (MAPK) pathway. Snail1 is a key transcription factor, induced by TGFβ that promotes migration and invasion of cancer cells. In this study, we have identified a novel binding site for c-Jun in the promoter of the Snail1 gene and report that the activation of the TGFβ-TRAF6-p38 MAPK pathway promotes both c-Jun expression and its activation via p38α-dependent phosphorylation of c-Jun at Ser63. The TRAF6-dependent activation of p38 also leads to increased stability of c-Jun, due to p38-dependent inactivation of glycogen synthase kinase (GSK) 3β by phosphorylation at Ser9. Thus, our findings elucidate a novel role for the p38 MAPK pathway in stimulated cells, leading to activation of c-Jun and its binding to the promoter of Snail1, thereby triggering motility and invasiveness of aggressive human prostate cancer cells.
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http://dx.doi.org/10.4161/cc.29339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4128885PMC
August 2015

Regulated intramembrane proteolysis of the TGFβ type I receptor conveys oncogenic signals.

Future Oncol 2014 Aug 5;10(11):1853-61. Epub 2014 Mar 5.

Department of Medical Biosciences, Pathology, Umeå University, SE-901 85 Umeå, Sweden.

Cancer cells produce high levels of TGFβ, a multipotent cytokine. Binding of TGFβ to its cell surface receptors, the transmembrane serine/threonine kinases TβRII and TβRI, causes phosphorylation and activation of intracellular latent Smad transcription factors. Nuclear Smads act in concert with specific transcription factors to reprogram epithelial cells to become invasive mesenchymal cells. TGFβ also propagates non-canonical signals, so it is crucial to have a better understanding of the underlying molecular mechanisms which favor this pathway. Here we highlight our recent discovery that TGFβ promotes the proteolytic cleavage of TβRI in cancer cells, resulting in the liberation and nuclear translocation of its intracellular domain, acting as co-regulator to transcribe pro-invasive genes. This newly identified oncogenic TGFβ pathway resembles the Notch signaling pathway. We discuss our findings in relation to Notch and provide a short overview of other growth factors that transduce signals via nuclear translocation of their cell surface receptors.
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http://dx.doi.org/10.2217/fon.14.45DOI Listing
August 2014

TRAF6 stimulates the tumor-promoting effects of TGFβ type I receptor through polyubiquitination and activation of presenilin 1.

Sci Signal 2014 Jan 7;7(307):ra2. Epub 2014 Jan 7.

1Department of Medical Biosciences, Pathology, Umeå University, SE-901 85 Umeå, Sweden.

Transforming growth factor-β (TGFβ) can be both a tumor promoter and suppressor, although the mechanisms behind the protumorigenic switch remain to be fully elucidated. The TGFβ type I receptor (TβRI) is proteolytically cleaved in the ectodomain region. Cleavage requires the combined activities of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and TNF-α-converting enzyme (TACE). The cleavage event occurs selectively in cancer cells and generates an intracellular domain (ICD) of TβRI, which enters the nucleus to mediate gene transcription. Presenilin 1 (PS1), a γ-secretase catalytic core component, mediates intramembrane proteolysis of transmembrane receptors, such as Notch. We showed that TGFβ increased both the abundance and activity of PS1. TRAF6 recruited PS1 to the TβRI complex and promoted lysine-63-linked polyubiquitination of PS1, which activated PS1. Furthermore, PS1 cleaved TβRI in the transmembrane domain between valine-129 and isoleucine-130, and ICD generation was inhibited when these residues were mutated to alanine. We also showed that, after entering the nucleus, TβRI-ICD bound to the promoter and increased the transcription of the gene encoding TβRI. The TRAF6- and PS1-induced intramembrane proteolysis of TβRI promoted TGFβ-induced invasion of various cancer cells in vitro. Furthermore, when a mouse xenograft model of prostate cancer was treated with the γ-secretase inhibitor DBZ {(2S)-2-[2-(3,5-difluorophenyl)-acetylamino]-N-(5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)-propionamide}, generation of TβRI-ICD was prevented, transcription of the gene encoding the proinvasive transcription factor Snail1 was reduced, and tumor growth was inhibited. These results suggest that γ-secretase inhibitors may be useful for treating aggressive prostate cancer.
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http://dx.doi.org/10.1126/scisignal.2004207DOI Listing
January 2014

Non-Smad signaling pathways.

Cell Tissue Res 2012 Jan 24;347(1):11-20. Epub 2011 Jun 24.

Medical Biosciences, Umeå University, SE-901 85 Umeå, Sweden.

Transforming growth factor-beta (TGFβ) is a key regulator of cell fate during embryogenesis and has also emerged as a potent driver of the epithelial-mesenchymal transition during tumor progression. TGFβ signals are transduced by transmembrane type I and type II serine/threonine kinase receptors (TβRI and TβRII, respectively). The activated TβR complex phosphorylates Smad2 and Smad3, converting them into transcriptional regulators that complex with Smad4. TGFβ also uses non-Smad signaling pathways such as the p38 and Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways to convey its signals. Ubiquitin ligase tumor necrosis factor (TNF)-receptor-associated factor 6 (TRAF6) and TGFβ-associated kinase 1 (TAK1) have recently been shown to be crucial for the activation of the p38 and JNK MAPK pathways. Other TGFβ-induced non-Smad signaling pathways include the phosphoinositide 3-kinase-Akt-mTOR pathway, the small GTPases Rho, Rac, and Cdc42, and the Ras-Erk-MAPK pathway. Signals induced by TGFβ are tightly regulated and specified by post-translational modifications of the signaling components, since they dictate the subcellular localization, activity, and duration of the signal. In this review, we discuss recent findings in the field of TGFβ-induced responses by non-Smad signaling pathways.
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http://dx.doi.org/10.1007/s00441-011-1201-yDOI Listing
January 2012

TRAF6 ubiquitinates TGFβ type I receptor to promote its cleavage and nuclear translocation in cancer.

Nat Commun 2011 ;2:330

Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, SE-751 85 Uppsala, Sweden.

Transforming growth factor β (TGFβ) is a pluripotent cytokine promoting epithelial cell plasticity during morphogenesis and tumour progression. TGFβ binding to type II and type I serine/threonine kinase receptors (TβRII and TβRI) causes activation of different intracellular signaling pathways. TβRI is associated with the ubiquitin ligase tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6). Here we show that TGFβ, via TRAF6, causes Lys63-linked polyubiquitination of TβRI, promoting cleavage of TβRI by TNF-alpha converting enzyme (TACE), in a PKCζ-dependent manner. The liberated intracellular domain (ICD) of TβRI associates with the transcriptional regulator p300 to activate genes involved in tumour cell invasiveness, such as Snail and MMP2. Moreover, TGFβ-induced invasion of cancer cells is TACE- and PKCζ- dependent and the TβRI ICD is localized in the nuclei of different kinds of tumour cells in tissue sections. Thus, our data reveal a specific role for TβRI in TGFβ mediated tumour invasion.
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http://dx.doi.org/10.1038/ncomms1332DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3113296PMC
September 2011
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