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TRAF6 stimulates the tumor-promoting effects of TGF beta type I receptor through polyubiquitination and activation of Presenilin 1
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
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2014 (English)In: Science Signaling, ISSN 1945-0877, E-ISSN 1937-9145, Vol. 7, no 307, ra2Article in journal (Refereed) Published
Abstract [en]

Transforming growth factor-beta (TGF beta) can be both a tumor promoter and suppressor, although the mechanisms behind the protumorigenic switch remain to be fully elucidated. The TGF beta type I receptor (T beta RI) is proteolytically cleaved in the ectodomain region. Cleavage requires the combined activities of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and TNF-alpha-converting enzyme (TACE). The cleavage event occurs selectively in cancer cells and generates an intracellular domain (ICD) of T beta RI, which enters the nucleus to mediate gene transcription. Presenilin 1 (PS1), a gamma-secretase catalytic core component, mediates intramembrane proteolysis of transmembrane receptors, such as Notch. We showed that TGF beta increased both the abundance and activity of PS1. TRAF6 recruited PS1 to the T beta RI complex and promoted lysine-63-linked polyubiquitination of PS1, which activated PS1. Furthermore, PS1 cleaved T beta RI 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, T beta RI-ICD bound to the promoter and increased the transcription of the gene encoding T beta RI. The TRAF6- and PS1-induced intramembrane proteolysis of T beta RI promoted TGF beta-induced invasion of various cancer cells in vitro. Furthermore, when a mouse xenograft model of prostate cancer was treated with the gamma-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 T beta RI-ICD was prevented, transcription of the gene encoding the proinvasive transcription factor Snail1 was reduced, and tumor growth was inhibited. These results suggest that gamma-secretase inhibitors may be useful for treating aggressive prostate cancer.

Place, publisher, year, edition, pages
American Association for the Advancement of Science , 2014. Vol. 7, no 307, ra2
National Category
Biochemistry and Molecular Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:umu:diva-85774DOI: 10.1126/scisignal.2004207ISI: 000329401100004OAI: oai:DiVA.org:umu-85774DiVA: diva2:696294
Funder
Swedish Research Council, K2010-67X-15284-01-3Swedish Cancer Society, 100303Knut and Alice Wallenberg Foundation, 2012.0090
Available from: 2014-02-13 Created: 2014-02-10 Last updated: 2017-12-06Bibliographically approved
In thesis
1. TRAF6 stimulates TGFβ-induced oncogenic signal transduction in cancer cells
Open this publication in new window or tab >>TRAF6 stimulates TGFβ-induced oncogenic signal transduction in cancer cells
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
TRAF6 stimulerar TGFβ-inducerad onkogen signal transduction i cancerceller.
Abstract [en]

Prostate cancer is one of the leading causes of cancer-related deaths in men worldwide, with 10,000 new cases/year diagnosed in Sweden. In this context, there is an urgent need to identify new biomarkers to detect prostate cancer at an initial stage for earlier treatment intervention. Although how prostate cancer develops has not been fully established, the male sex hormone testosterone is a known prerequisite for prostate cancer development. High levels of transforming growth factor-β (TGFβ) are prognostically unfavorable in prostate cancer patients.

TGFβ is a multifunctional cytokine that regulates a broad range of cellular responses. TGFβ signals through either the canonical Smad or the non-Smad signaling cascade. Cancerous cells develop different strategies to evade defense mechanisms and metastasize to different parts of the body. This thesis unveils one such novel mechanism related to TGFβ signaling.

The first two articles provide evidence that TGFβ receptor type I (TβRI) is ubiquitinated by tumor necrosis factor receptor-associated factor 6 (TRAF6) and is cleaved at the ectodomain region by tumor necrosis factor alpha converting enzyme (TACE) in a protein kinase C zeta type-dependent manner. After TβRI is shed from the ectodomain, it undergoes a second cleavage by presenilin 1 (PS1), a γ-secretase catalytic subunit, which liberates the TβRI intracellular domain (TβRI-ICD) from the cell membrane. TRAF6 promotes TGFβ-dependent Lys63-linked polyubiquitination and recruitment of PS1 to the TβRI complex, and facilitates the cleavage of TβRI by PS1 to generate a TβRI-ICD. The TβRI-ICD then translocates to the nucleus, where it binds with the transcriptional co-activator p300 and regulates the transcription of pro-invasive target genes such as Snail1. Moreover, the nuclear translocated TβRI-ICD cooperates with the Notch intracellular domain (NICD), a core component in the Notch signaling pathway, to drive the expression of invasive genes. Interestingly, treatment with g-secretase inhibitors was able to inhibit cleavage of TβRI and inhibit the TGFβ-induced oncogenic pathway in an in vivo prostate cancer xenograft model.

In the third article, we identified that Lysine 178 is the acceptor lysine in TβRI that is ubiquitinated by TRAF6. The TβRI K178R mutant was neither ubiquitinated nor translocated to the nucleus, and prevented transcriptional regulation of invasive genes in a dominant negative manner.

In the fourth article, we show that TGFβ utilizes the E3-ligase TRAF6 and the p38 mitogen-activated protein kinase to phosphorylate c-Jun. In turn, the phosphorylated c-Jun activates p21 and Snail1 in a non-canonical Smad-independent pathway, and thereby promotes invasion in cancerous cells.

In summary, we elucidate a new mechanism of TGFβ-induced oncogenic signal transduction in cancer cells in which TRAF6 plays a fundamental role. This opens a new avenue in the field of TGFβ signaling.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2014. 70 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1638
Keyword
c-Jun, invasion, non-Smad, Notch, NICD, oncogenesis, presenilin1, PKCζ, prostate cancer, p21, p38, p300, Snail1, TGFβ, TβRI, TACE, TRAF6, ubiquitination, TGFbeta signal transduction
National Category
Cancer and Oncology
Research subject
Pathology
Identifiers
urn:nbn:se:umu:diva-87017 (URN)978-91-7601-025-9 (ISBN)
Public defence
2014-04-11, Hörsal E 04, Unod R1, Norrlands universitetssjukhus, Umeå, 10:15 (English)
Opponent
Supervisors
Funder
Swedish Cancer SocietySwedish Research Council
Available from: 2014-03-19 Created: 2014-03-18 Last updated: 2014-03-19Bibliographically approved
2. TRAF6, a key regulator of TGFβ-induced oncogenesis in prostate cancer
Open this publication in new window or tab >>TRAF6, a key regulator of TGFβ-induced oncogenesis in prostate cancer
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Prostate cancer is the most common cancer in men, with the incidence rapidly increasing in Europe over the past two decades. Reliable biomarkers for prostate cancer are currently unavailable. Thus, there is an urgent need for improved biomarkers to diagnose prostate cancer at an early stage and to determine the best treatment options. Higher expression of transforming growth factor-β (TGFβ) has been reported in patients with aggressive cancer.

TGFβ is a multifunctional cytokine that acts as a tumor suppressor during early tumor development, and as a tumor promoter at later stages of cancer. TGFβ signals through the canonical Smad or non-Smad cascade via TGFβ type II and type I receptors. The TGFβ signaling cascade is regulated by various post-translational modifications of its key components. The present investigation aimed to identify a potential function of TRAF6 in TGFβ-induced responses in prostate cancer.

The first two articles of this thesis unveil the proteolytic cleavage of TGFβ type I receptor (TβRI), and the biological importance of the liberated TβRI intracellular domain (TβRI-ICD) in the nucleus. We found that tumor necrosis factor receptor-associated factor 6 (TRAF6) polyubiquitinates TβRI, which leads to cleavage of TβRI by tumor necrosis factor alpha converting enzyme (TACE) in a protein kinase C zeta (PKCζ)-dependent manner. Following ectodomain shedding, TβRI undergoes a second cleavage by presenilin 1 (PS1), which liberates TβRI-ICD. TβRI-ICD translocates to the nucleus, where it regulates its own expression as well as expression of the pro-invasive gene Snail1, thereby promoting invasion. We further found that TβRI-ICD associates with Notch intracellular domain (NICD) to drive expression of the pro-invasive gene Snail1, as well as Notch1 ligand Jag1.

The third article provides evidence that TRAF6 promotes Lys63-linked polyubiquitination of TβRI at Lys178 in a TGFβ-dependent manner. TβRI polyubiquitination was found to be a prerequisite for TβRI nuclear translocation, and thus for regulation of the genes involved in cell cycle, differentiation, and invasion of prostate cancer cells.

In the fourth article we investigated the role of the pro-invasive gene Snail1 in TGFβ-induced epithelial-to-mesenchymal transition (EMT) in prostate cancer cells.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2015. 55 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1739
Keyword
TβRI, TGFβ, TACE, TRAF6, PS1, PKCζ, TβRI-ICD, NICD, Smad, non-Smad, prostate cancer, Snail1, MMP, p300, p21, PAI1, ubiquitination, cleavage, ICD, invasion, HES1, signaling
National Category
Cell and Molecular Biology
Research subject
Pathology
Identifiers
urn:nbn:se:umu:diva-108014 (URN)978-91-7601-315-1 (ISBN)
Public defence
2015-09-25, Hörsal Betula, Norrlands Universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Funder
Swedish Cancer SocietyThe Kempe FoundationsKnut and Alice Wallenberg Foundation
Available from: 2015-09-04 Created: 2015-09-01 Last updated: 2015-09-03Bibliographically approved

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