Inactivation of the Tumor Suppressor CYLD Sensitizes Mice to Breast Cancer Development.

BACKGROUND/AIM: Breast cancer is a leading cause of cancer-related deaths among women. Down-regulation of the tumor suppressor gene Cyld in breast cancer has been linked to a poor prognosis. This study investigated the role of Cyld in breast cancer using conditional mutant mouse models carrying a Cyld mutation, which inactivates the deubiquitinating activity of its protein product CYLD in mammary epithelial cells. MATERIALS AND METHODS: We examined the potential of CYLD inactivation to induce mammary tumors spontaneously or modify the susceptibility of mice to mammary tumorigenesis by DMBA treatment or ErbB2 over-expression. RESULTS: CYLD inactivation significantly increased susceptibility to breast cancer induced by either DMBA treatment or ErbB2 over-expression. Moreover, while CYLD inactivation alone did not lead to spontaneous mammary tumorigenesis, it did contribute to the formation of multifocal hyperplastic lesions in virgin mice of predominantly FVB/NJ background. CONCLUSION: Our study demonstrates the tumor enhancing potential of CYLD inactivation in mammary tumorigenesis in vivo and establishes novel relevant mouse models that can be exploited for developing prognostic and therapeutic protocols.

The Tumor Suppressor CYLD Inhibits Mammary Epithelial to Mesenchymal Transition by the Coordinated Inhibition of YAP/TAZ and TGF Signaling.

Downregulation of the cylindromatosis (CYLD) tumor suppressor has been associated with breast cancer development and progression. Here, we report a critical role for CYLD in maintaining the phenotype of mammary epithelial cells in vitro and in vivo. CYLD downregulation or inactivation induced an epithelial to mesenchymal transition of mammary epithelial cells that was dependent on the concomitant activation of the transcription factors Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) and transforming growth factor beta (TGF)signaling. CYLD inactivation enhanced the nuclear localization of YAP/TAZ and the phosphorylation of Small Mothers Against Decapentaplegic (SMAD)2/3 proteins in confluent cell culture conditions. Consistent with these findings were the hyperplastic alterations of CYLD-deficient mouse mammary epithelia, which were associated with enhanced nuclear expression of the YAP/TAZ transcription factors. Furthermore, in human breast cancer samples, downregulation of CYLD expression correlates with enhanced YAP/TAZ-regulated target gene expression. Our results identify CYLD as a critical regulator of a signaling node that prevents the coordinated activation of YAP/TAZ and the TGF pathway in mammary epithelial cells, in order to maintain their phenotypic identity and homeostasis. Consequently, they provide a novel conceptual framework that supports and explains a causal implication of deficient CYLD expression in aggressive human breast cancers.

Systemic therapy for chromophobe renal cell carcinoma: A systematic review.

BACKGROUND: Chromophobe renal cell carcinoma (chRCC) subtype accounts for almost 5% of total RCC cases. It carries the best prognosis among the rest of RCC types. However, patients with metastatic chRCC disease have worse prognosis than patients with advanced clear cell RCC. Furthermore, available data regarding systemic therapy for chRCC patients are scarce and confusing. AIM: The aim of this systematic review is to search for and critically appraise studies that investigate the results of systemic therapies in patients diagnosed with metastatic chRCC disease. METHODS: Search strategy included PUBMED, CENTRAL, clinicaltrials.gov databases, and abstracts of major conferences with a focus on urologic oncology (till March 2019). Studies investigating patients that were treated with systemic therapy for advanced chRCC disease were included. Primary outcomes were progression-free survival and objective response rate. Secondary outcome was overall survival. Screening of available studies was carried out by 2 groups of reviewers, as well as the quality assessment of the included studies. RESULTS: The systematic search yielded 369 studies, of which 15 studies (2 randomized control trials and 13 cohort studies), involving 183 patients, met the eligibility criteria. The 2 randomized control trials that directly compared sunitinib vs. everolimus, suggested an advantage for sunitinib without being statistically significant. Furthermore, sunitinib seems to be superior than sorafenib at least in terms of objective response rate. Regarding mTOR inhibitors, they may have a role in a specific subset of chRCC patients, that needs to be further explored. Finally, as far as immunotherapy is concerned, available data suggest that chRCC seems to be resistant to recent immune check point inhibitors, since just a few tumor responses were observed with the administered immunotherapy regiments. CONCLUSION: The optimum therapy for metastatic chRCC is still missing, as results from ongoing trials are awaited. More studies, of high quality and adequate sample size, that will be based on the specific biology of chRCC, have to be carried out in order to identify the best treatment.

Functional analysis of the C. elegans cyld-1 gene reveals extensive similarity with its human homolog.

The human cylindromatosis tumor suppressor (HsCyld) has attracted extensive attention due to its association with the development of multiple types of cancer. HsCyld encodes a deubiquitinating enzyme (HsCYLD) with a broad range of functions that include the regulation of several cell growth, differentiation and death pathways. HsCyld is an evolutionarily conserved gene. Homologs of HsCyld have been identified in simple model organisms such as Drosophila melanogaster and Caenorhabditis elegans (C. elegans) which offer extensive possibilities for functional analyses. In the present report we have investigated and compared the functional properties of HsCYLD and its C. elegans homolog (CeCYLD). As expected from the mammalian CYLD expression pattern, the CeCyld promoter is active in multiple tissues with certain gastrointestinal epithelia and neuronal cells showing the most prominent activity. CeCYLD is a functional deubiquitinating enzyme with similar specificity to HsCYLD towards K63- and M1-linked polyubiquiting chains. CeCYLD was capable of suppressing the TRAF2-mediated activation of NF-kappaB and AP1 similarly to HsCYLD. Finally, CeCYLD could suppress the induction of TNF-dependent gene expression in mammalian cells similarly to HsCYLD. Our results demonstrate extensively overlapping functions between the HsCYLD and CeCYLD, which establish the C. elegans protein as a valuable model for the elucidation of the complex activity of the human tumor suppressor protein.

Down-regulation of the Tumor Suppressor CYLD Enhances the Transformed Phenotype of Human Breast Cancer Cells.

BACKGROUND/AIM: The cylindromatosis tumor suppressor (CYLD) has been implicated in the inhibition of human breast cancer development by virtue of the poor prognosis of patients with down-regulated CYLD expression. In order to investigate the mechanism of breast cancer suppression by CYLD, in the present study, cellular and molecular aspects of CYLD-dependent phenotypic regulation of different types of human breast cancer cell lines were analyzed. MATERIALS AND METHODS: CYLD expression was down-regulated by RNA interference in human breast cancer cell lines. Parental and CYLD-deficient cell lines were evaluated for their viability, migratory capacity, anchorage-independent growth and chemoresistance. Wild-type and mutated forms of CYLD were also evaluated for their ability to suppress the clonogenic potential of breast cancer cells. RESULTS: CYLD down-regulation enhanced the survival and migratory properties of basal and luminal breast cancer cell lines. In addition, down-regulation of CYLD expression enhanced the ability of human breast cancer cells to grow in an anchorage-independent manner and could be associated with resistance to chemotherapeutic drugs. The growth-suppressive properties of CYLD on breast cancer cell lines were dependent on its de-ubiquitinating activity and its amino terminal cytoskeleton-interacting region. CONCLUSION: Our results establish a broad range of tumor-suppressive properties that are conferred by CYLD in basal and luminal human breast cancer cells and support the significance of targeted de-ubiquitination by CYLD in breast cancer cell growth suppression.

Inactivation of CYLD in intestinal epithelial cells exacerbates colitis-associated colorectal carcinogenesis - a short report.

PURPOSE: CYLD is a tumor suppressor that has been linked to the development of various human malignancies, including colon cancer. The tumor-suppressing function of CYLD is associated with its deubiquitinating activity, which maps to the carboxyl-terminal region of the protein. In the present study we evaluated the role of intestinal epithelial CYLD in colitis-associated cancer using a conditional mouse CYLD inactivation model. METHODS: In order to evaluate the role of CYLD in intestinal epithelial carcinogenesis, mice (IEC-Cyld (Δ9) mice) that carry a mutation that eliminates the deubiquitinating domain of CYLD in intestinal epithelial cells (IEC) were generated by crossing Villin-Cre transgenic mice to previously generated mice carrying a loxP-flanked Cyld exon 9 (Cyld (flx9) mice). RESULTS: We found that IEC-Cyld (Δ9) mice did not present spontaneous intestinal abnormalities up to one year of age. However, upon challenge with a combination of genotoxic (AOM) and pro-inflammatory (DSS) agents we found that the number of adenomas in the IEC-Cyld (Δ9) mice was dramatically increased compared to the control mice. Inactivation of CYLD in intestinal epithelial cells did not affect the classical nuclear factor-kappaB (NF-κB) and c-Jun kinase (JNK) activation pathways under physiological conditions, suggesting that these pathways do not predispose CYLD-deficient intestinal epithelia to colorectal cancer development before the onset of genotoxic and/or pro-inflammatory stress. CONCLUSIONS: Our findings underscore a critical tumor-suppressing role for functional intestinal epithelial CYLD in colitis-associated carcinogenesis. CYLD expression and its associated pathways in intestinal tumors may be exploited for future prognostic and therapeutic purposes.

The PP4R1 subunit of protein phosphatase PP4 targets TRAF2 and TRAF6 to mediate inhibition of NF-κB activation.

TRAFs constitute a family of proteins that have been implicated in signal transduction by immunomodulatory cellular receptors and viral proteins. TRAF2 and TRAF6 have an E3-ubiquitin ligase activity, which is dependent on the integrity of their RING finger domain and it has been associated with their ability to activate the NF-κB and AP1 signaling pathways. A yeast two-hybrid screen with TRAF2 as bait, identified the regulatory subunit PP4R1 of protein phosphatase PP4 as a TRAF2-interacting protein. The interaction of TRAF2 with PP4R1 depended on the integrity of the RING finger domain of TRAF2. PP4R1 could interact also with the TRAF2-related factor TRAF6 in a RING domain-dependent manner. Exogenous expression of PP4R1 inhibited NF-κB activation by TRAF2, TRAF6, TNF and the Epstein-Barr virus oncoprotein LMP1. In addition, expression of PP4R1 downregulated IL8 induction by LMP1, whereas downregulation of PP4R1 by RNA interference enhanced the induction of IL8 by LMP1 and TNF. PP4R1 could mediate the dephosphorylation of TRAF2 Ser11, which has been previously implicated in TRAF2-mediated activation of NF-κB. Finally, PP4R1 could inhibit TRAF6 polyubiquitination, suggesting an interference with the E3 ubiquitin ligase activity of TRAF6. Taken together, our data identify a novel mechanism of NF-κB pathway inhibition which is mediated by PP4R1-dependent targeting of specific TRAF molecules.

Mutational analysis of TRAF6 reveals a conserved functional role of the RING dimerization interface and a potentially necessary but insufficient role of RING-dependent TRAF6 polyubiquitination towards NF-κB activation.

TRAF6 is an E3 ubiquitin ligase that plays a pivotal role in the activation of NF-κB by innate and adaptive immunity stimuli. TRAF6 consists of a highly conserved carboxyl terminal TRAF-C domain which is preceded by a coiled coil domain and an amino terminal region that contains a RING domain and a series of putative zinc-finger motifs. The TRAF-C domain contributes to TRAF6 oligomerization and mediates the interaction of TRAF6 with upstream signaling molecules whereas the RING domain comprises the core of the ubiquitin ligase catalytic domain. In order to identify structural elements that are important for TRAF6-induced NF-κB activation, mutational analysis of the TRAF-C and RING domains was performed. Alterations of highly conserved residues of the TRAF-C domain of TRAF6 did not affect significantly the ability of the protein to activate NF-κB. On the other hand a number of functionally important residues (L77, Q82, R88, F118, N121 and E126) for the activation of NF-κB were identified within the RING domain of TRAF6. Interestingly, several homologues of these residues in TRAF2 were shown to have a conserved functional role in TRAF2-induced NF-κB activation and lie at the dimerization interface of the RING domain. Finally, whereas alteration of Q82, R88 and F118 compromised both the K63-linked polyubiquitination of TRAF6 and its ability to activate NF-κB, alteration of L77, N121 and E126 diminished the NF-κB activating function of TRAF6 without affecting TRAF6 K63-linked polyubiquitination. Our results support a conserved functional role of the TRAF RING domain dimerization interface and a potentially necessary but insufficient role for RING-dependent TRAF6 K63-linked polyubiquitination towards NF-κB activation in cells.

Constitutive CD40 signaling phenocopies the transforming function of the Epstein-Barr virus oncoprotein LMP1 in vitro.

The oncoprotein LMP1 mimics an activated CD40 receptor, yet it is not known whether constitutive CD40 signaling, like LMP1, is sufficient to transform cells. Here we demonstrate that constitutive activation of the CD40 pathway by a chimeric LMP1CD40 molecule resembles the transforming function of LMP1 in inducing loss of contact inhibition and anchorage independent growth of Rat1 fibroblasts. Rat1 transformation correlates with the expression level of LMP1CD40 and depends on its ability to oligomerize. Our data provide direct evidence for the oncogenic potential of the CD40 signaling pathway, which is also established as a model-mechanism for LMP1-induced transformation.

Human ubiquitin specific protease 31 is a deubiquitinating enzyme implicated in activation of nuclear factor-kappaB.

TRAF2 mediates activation of the transcription factors NF-kappaB and AP1 by TNF. A yeast two-hybrid screen of a human cDNA library identified a ubiquitin specific protease homologue (USP31) as a TRAF2-interacting protein. Two cDNAs encoding for USP31 were identified. One cDNA encodes a 1035-amino acid long isoform of USP31 (USP31, long isoform) and the other a 485-amino acid long isoform of USP31 (USP31S1, short isoform). USP31 and USP31S1 share a common amino terminal region with homology to the catalytic region of known deubiquitinating enzymes. Enzymatic assays demonstrated that USP31 but not USP31S1 possess deubiquitinating activity. Furthermore, it was shown that USP31 has a higher activity towards lysine-63-linked as compared to lysine-48-linked polyubiquitin chains. Overexpression of USP31 in HEK 293T cells inhibited TNFalpha, CD40, LMP1, TRAF2, TRAF6 and IKKbeta-mediated NF-kappaB activation, but did not inhibit Smad-mediated transcription activation. In addition, both USP31 isoforms interact with p65/RelA. Our data support a role for USP31 in the regulation of NF-kappaB activation by members of the TNF receptor superfamily.

Induction of apoptosis by rewiring the signal transduction of Epstein-Barr virus oncoprotein LMP1 toward caspase activation.

The Epstein-Barr virus latent membrane protein 1 (LMP1) is an oncoprotein which mimics activated tumor necrosis factor receptor family members. Here we demonstrate the principle that an inducible association of the LMP1 cytoplasmic carboxyl terminus with caspase-8 by a heterodimerizing agent causes apoptosis. This process depends on the catalytic activity of caspase-8 and the ability of LMP1 to oligomerize constitutively at the plasma membrane. Our data indicate that chemical inducers of the association of the LMP1 carboxyl terminus with caspase-8 can kill LMP1-expressing cells selectively. Such compounds could be used as chemotherapeutic agents for LMP1-associated malignancies.

Functional specificity of two hormone response elements present on the human apoA-II promoter that bind retinoid X receptor alpha/thyroid receptor beta heterodimers for retinoids and thyroids: synergistic interactions between thyroid receptor beta and upstream stimulatory factor 2a.

DNA binding and mutagenesis in vitro established that the -67/-55 region of the apoA-II (apolipoprotein A-II) promoter contains a thyroid HRE (hormone response element), which strongly binds RXRalpha (retinoid X receptor alpha)/T(3)Rbeta (thyroid receptor beta) heterodimers and weakly T(3)Rbeta homodimers, but does not bind other homo- or heterodimers of RXRalpha or orphan nuclear receptors. Transactivation was abolished by point mutations in the thyroid HRE. In co-transfection experiments of HEK-293 (human embryonic kidney 293) cells, the -911/+29 human apoA-II promoter was transactivated strongly by RXRalpha/T(3)Rbeta heterodimers in the presence of RA (9- cis retinoic acid) or T(3) (tri-iodothyronine). Homopolymeric promoters containing either three copies of the -73/-40 (element AIIAB) or four copies of the -738/-712 (element AIIJ) apoA-II promoter could be transactivated by RXRalpha/T(3)Rbeta heterodimers in COS-7 cells only in the presence of T(3) or RA respectively. RXRalpha/T(3)Rbeta heterodimers and USF2a (upstream stimulatory factor 2a) synergistically transactivated the -911/+29 apoA-II promoter in the presence of T(3). USF2a also enhanced the activity of a GAL4-T(3)Rbeta fusion protein in the presence of T(3) and suppressed the activity of a GAL4-RXRalpha fusion protein in the presence of RA. These findings suggest a functional specificity of the two HREs of the apoA-II promoter for retinoids and thyroids, which is modulated by synergistic or antagonistic interactions between RXRalpha/T(3)Rbeta heterodimers and the ubiquitous transcription factor USF2a.

CYLD is a deubiquitinating enzyme that negatively regulates NF-kappaB activation by TNFR family members.

Familial cylindromatosis is an autosomal dominant predisposition to tumours of skin appendages called cylindromas. Familial cylindromatosis is caused by mutations in a gene encoding the CYLD protein of previously unknown function. Here we show that CYLD is a deubiquitinating enzyme that negatively regulates activation of the transcription factor NF-kappaB by specific tumour-necrosis factor receptors (TNFRs). Loss of the deubiquitinating activity of CYLD correlates with tumorigenesis. CYLD inhibits activation of NF-kappaB by the TNFR family members CD40, XEDAR and EDAR in a manner that depends on the deubiquitinating activity of CYLD. Downregulation of CYLD by RNA-mediated interference augments both basal and CD40-mediated activation of NF-kappaB. The inhibition of NF-kappaB activation by CYLD is mediated, at least in part, by the deubiquitination and inactivation of TNFR-associated factor 2 (TRAF2) and, to a lesser extent, TRAF6. These results indicate that CYLD is a negative regulator of the cytokine-mediated activation of NF-kappaB that is required for appropriate cellular homeostasis of skin appendages.

Cellular signaling pathways engaged by the Epstein-Barr virus transforming protein LMP1.

The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is an essential component of the viral machinery that orchestrates cellular transformation and oncogenesis. The critical role of LMP1 in transformation has been established through recombinant genetic analysis of the EBV genome, ectopic expression in cell lines and transgenic mice and immunohistochemical analysis of EBV- associated tumor specimens. The principal mechanism of LMP1 function is based on mimicry of activated cell surface receptors of the tumor necrosis factor superfamily. LMP1 signaling culminates in the activation of transcription factors NF-kappaB, AP1 and STAT1/3, which have been tightly linked to prevention of apoptosis and malignant transformation. The molecular mechanisms of LMP1 function will be reviewed in this report.