Component Of Ubiquitin Ligase Research Articles

The Deubiquitinase USP46 Is Essential for Proliferation and Tumor Growth of HPV-Transformed Cancers

High-risk human papilloma viruses (HPVs) cause cervical, anal, and oropharyngeal cancers, unlike the low-risk HPVs, which cause benign lesions. E6 oncoproteins from the high-risk strains are essential for cell proliferation and transformation in HPV-induced cancers. We report that a cellular deubiquitinase, USP46, is selectively recruited by the E6 of high-risk, but not low-risk, HPV to deubiqutinate and stabilize Cdt2/DTL. Stabilization of Cdt2, a component of the CRL4Cdt2 E3 ubiquitin ligase, limits the level of Set8, an epigenetic writer, and promotes cell proliferation. USP46 is essential for the proliferation of HPV-transformed cells, but not of cells without HPV. Cdt2 is elevated in human cervical cancers and knockdown of USP46 inhibits HPV-transformed tumor growth in xenografts. Recruitment of a cellular deubiquitinase to stabilize key cellular proteins is animportant activity of oncogenic E6, and the importance of E6-USP46-Cdt2-Set8 pathway in HPV-induced cancers makes USP46 a target for the therapy of such cancers.

Abstract 068: Kelch-Like 3 Dephosphorylation by Calcineurin as a Mechanism Regulating Na-cl Cotransporter

Tacrolimus, an inhibitor of the phosphatase calcineurin, is widely used as an immunosuppressive agent. However, its use is associated with hypertension and hyperkalemia. Although previous studies indicated that the activation of thiazide-sensitive, Na-Cl cotransporter (NCC) plays a key role in the pathogenesis, the identity of the direct target of calcineurin in kidney remained unclear. Kelch-like 3 (KLHL3) is a component of an E3 ubiquitin ligase that critically regulates NCC function. In pseudophyoaldoseronism type 2, the disease-causing mutations in KLHL3 impair binding and degradation of with-no-lysine 1 (WNK1) and WNK4, resulting in hypertension and hyperkalemia that are correctable by thiazide. Previously, we reported that phosphorylation of KLHL3 at serine 433 (KLHL3 S433-P ) by protein kinase C critically regulates KLHL3 function by abrogating the substrate-binding ability. However, phosphatases responsible for KLHL3 S433-P regulation were unknown. We here show that calcineurin acts as a phosphatase for KLHL3 S433-P . In in vitro phosphatase assay, we found that calcineurin suppressed KLHL3 S433-P levels (65% reduction from baseline). Consistently, the activation of calcineurin by ionomycin significantly reduced KLHL3 S433-P abundance in distal convoluted tubule cells and in HEK cells expressing KLHL3, an effect that was almost completely prevented by calcineurin knockdown. Moreover, we found that tacrolimus increased KLHL3 S433-P levels by 1.8 fold in mouse kidney, which was associated with the WNK accumulation and increased NCC activity. Finally, we found that tacrolimus attenuated KLHL3-mediated WNK4 ubiquitination and degradation, and this effect was abolished by non-phosphorylatable S433A substitution in KLHL3. These data identify Kelch-like 3 as a direct target of calcineurin, and provide evidence that KLHL3 is involved in the pathophysiology of tacrolimus-induced hypertension.

GAMBLING DISORDER: PERSONALITY CHARACTERISTICS IN A CLINICAL SAMPLE

Clear cell renal cell carcinoma (cc-RCC), is a serious cancer regarding; its fatality, liability for metastases and chemoresistance, so identification of recent therapeutic targets to improve the patients prognosis is needed. SPOP is a BTB/POZ domain containing speckle-type POZ protein, has been identified as an E3 ubiquitin ligase component. ZEB1 is an essential epithelial mesenchymal transition (EMT) activator; E-cadherin is a cell adhesion protein that had been detected in normal epithelial cells membrane.Was to assess the tissue protein markers SPOP, ZEB1 & E-cadherin expressions in benign areas of neoplastic kidney specimens and in cc-RCC patients, then correlating their expression levels with patients clinicopathological and prognostic data.We evaluated SPOP, ZEB-1 & E-cadherin expression using immunohistochemistry in samples from 50 cc-RCC and 20 benign areas of neoplastic kidney specimens, then we followed our patients for 5 years and finally we have analyzed correlations between the levels of markers expressions with patients clinicopathological and prognostic criteria in cc-RCC.Positive expression of SPOP & ZEB1 in addition to negative E- cadherin expression was detected in cc-RCC more than benign areas of neoplastic kidney specimens (p = 0.004 and p < 0.001 respectively). In cc-RCC Positive expression of SPOP, ZEB1 and negative E- cadherin expression was associated with higher grade (p = 0.006, 0.007 & <0.001 respectively), advanced AJCC stage (p = 0.013, 0.023 & <0.001 respectively), presence of L.N metastases (p = 0.002 = 0.010 and <0.001 respectively), distant metastases (p = 0.001, 0.003 & 0.035 respectively), poor PFS and OS rates (p < 0.001 and p = 0.013 respectively).Positive expression of SPOP& ZEB1 in addition to negative E- cadherin are associated with poor prognosis in cc-RCC patients.

Collaborating genomic, transcriptomic and microbiomic alterations lead to canine extreme intestinal polyposis

Extreme intestinal polyposis in pet dogs has not yet been reported in literature. We identified a dog patient who developed numerous intestinal polyps, with the severity resembling human classic familial adenomatous polyposis (FAP), except the jejunum-ileum junction being the most polyp-dense. We investigated this dog, in comparison with 22 other dogs with spontaneous intestinal tumors but no severe polyposis, and with numerous published human cancers. We found, not APC mutation, but three other alteration pathways as likely reasons of this canine extreme polyposis. First, somatic truncation mutation W411X of FBXW7, a component of an E3 ubiquitin ligase, over-activates MYC and cell cycle-promoting network, accelerating crypt cell proliferation. Second, genes of protein trafficking and localization are downregulated, likely associated with germline mutation G406D of STAMBPL1, a K63-deubiquitinase, and MYC network activation. This inhibits epithelial apical-basolateral polarity establishment, preventing crypt cell differentiation. Third, Bacteroides uniformis, a commensal gut anaerobe, thrives and expresses abundantly thioredoxin and nitroreductase. These bacterial products could reduce oxidative stress linked to host germline mutation R51X of CYB5RL, a cytochrome b5 reductase homologue, decreasing cell death. Our work emphasizes the close collaboration of alterations across the genome, transcriptome and microbiome in promoting tumorigenesis.

New insights into donor directionality of mating-type switching in Schizosaccharomyces pombe.

Mating-type switching in Schizosaccharomyces pombe entails programmed gene conversion events regulated by DNA replication, heterochromatin, and the HP1-like chromodomain protein Swi6. The whole mechanism remains to be fully understood. Using a gene deletion library, we screened ~ 3400 mutants for defects in the donor selection step where a heterochromatic locus, mat2-P or mat3-M, is chosen to convert the expressed mat1 locus. By measuring the biases in mat1 content that result from faulty directionality, we identified in total 20 factors required for donor selection. Unexpectedly, these included the histone H3 lysine 4 (H3K4) methyltransferase complex subunits Set1, Swd1, Swd2, Swd3, Spf1 and Ash2, the BRE1-like ubiquitin ligase Brl2 and the Elongator complex subunit Elp6. The mutant defects were investigated in strains with reversed donor loci (mat2-M mat3-P) or when the SRE2 and SRE3 recombination enhancers, adjacent to the donors, were deleted or transposed. Mutants in Set1C, Brl2 or Elp6 altered balanced donor usage away from mat2 and the SRE2 enhancer, towards mat3 and the SRE3 enhancer. The defects in these mutants were qualitatively similar to heterochromatin mutants lacking Swi6, the NAD+-dependent histone deacetylase Sir2, or the Clr4, Raf1 or Rik1 subunits of the histone H3 lysine 9 (H3K9) methyltransferase complex, albeit not as extreme. Other mutants showed clonal biases in switching. This was the case for mutants in the NAD+-independent deacetylase complex subunits Clr1, Clr2 and Clr3, the casein kinase CK2 subunit Ckb1, the ubiquitin ligase component Pof3, and the CENP-B homologue Cbp1, as well as for double mutants lacking Swi6 and Brl2, Pof3, or Cbp1. Thus, we propose that Set1C cooperates with Swi6 and heterochromatin to direct donor choice to mat2-P in M cells, perhaps by inhibiting the SRE3 recombination enhancer, and that in the absence of Swi6 other factors are still capable of imposing biases to donor choice.

SPOP, ZEB-1 and E-cadherin expression in clear cell renal cell carcinoma (cc-RCC): Clinicopathological and prognostic significance

BackgroundClear cell renal cell carcinoma (cc-RCC), is a serious cancer regarding; its fatality, liability for metastases and chemoresistance, so identification of recent therapeutic targets to improve the patients prognosis is needed. SPOP is a BTB/POZ domain containing speckle-type POZ protein, has been identified as an E3 ubiquitin ligase component. ZEB1 is an essential epithelial mesenchymal transition (EMT) activator; E-cadherin is a cell adhesion protein that had been detected in normal epithelial cells membrane. AimWas to assess the tissue protein markers SPOP, ZEB1 & E-cadherin expressions in benign areas of neoplastic kidney specimens and in cc-RCC patients, then correlating their expression levels with patients clinicopathological and prognostic data. MethodsWe evaluated SPOP, ZEB-1 & E-cadherin expression using immunohistochemistry in samples from 50 cc-RCC and 20 benign areas of neoplastic kidney specimens, then we followed our patients for 5 years and finally we have analyzed correlations between the levels of markers expressions with patients clinicopathological and prognostic criteria in cc-RCC. ResultsPositive expression of SPOP & ZEB1 in addition to negative E- cadherin expression was detected in cc-RCC more than benign areas of neoplastic kidney specimens (p = 0.004 and p < 0.001 respectively). In cc-RCC Positive expression of SPOP, ZEB1 and negative E- cadherin expression was associated with higher grade (p = 0.006, 0.007 & <0.001 respectively), advanced AJCC stage (p = 0.013, 0.023 & <0.001 respectively), presence of L.N metastases (p = 0.002 = 0.010 and <0.001 respectively), distant metastases (p = 0.001, 0.003 & 0.035 respectively), poor PFS and OS rates (p < 0.001 and p = 0.013 respectively). ConclusionPositive expression of SPOP& ZEB1 in addition to negative E- cadherin are associated with poor prognosis in cc-RCC patients.

Dependency of Heterochromatin Domains on Replication Factors.

Chromatin structure regulates both genome expression and dynamics in eukaryotes, where large heterochromatic regions are epigenetically silenced through the methylation of histone H3K9, histone deacetylation, and the assembly of repressive complexes. Previous genetic screens with the fission yeast Schizosaccharomyces pombe have led to the identification of key enzymatic activities and structural constituents of heterochromatin. We report here on additional factors discovered by screening a library of deletion mutants for silencing defects at the edge of a heterochromatic domain bound by its natural boundary—the IR-R+ element—or by ectopic boundaries. We found that several components of the DNA replication progression complex (RPC), including Mrc1/Claspin, Mcl1/Ctf4, Swi1/Timeless, Swi3/Tipin, and the FACT subunit Pob3, are essential for robust heterochromatic silencing, as are the ubiquitin ligase components Pof3 and Def1, which have been implicated in the removal of stalled DNA and RNA polymerases from chromatin. Moreover, the search identified the cohesin release factor Wpl1 and the forkhead protein Fkh2, both likely to function through genome organization, the Ssz1 chaperone, the Fkbp39 proline cis-trans isomerase, which acts on histone H3P30 and P38 in Saccharomyces cerevisiae, and the chromatin remodeler Fft3. In addition to their effects in the mating-type region, to varying extents, these factors take part in heterochromatic silencing in pericentromeric regions and telomeres, revealing for many a general effect in heterochromatin. This list of factors provides precious new clues with which to study the spatiotemporal organization and dynamics of heterochromatic regions in connection with DNA replication.

RNA sequencing reveals widespread transcriptome changes in a renal carcinoma cell line.

We used RNA sequencing (RNA-Seq) technology to investigate changes in the transcriptome profile in the Caki-1 clear cell renal cell carcinoma (ccRCC) cells, which overexpress monocyte chemoattractant protein-induced protein 1 (MCPIP1). RNA-Seq data showed changes in 11.6% and 41.8% of the global transcriptome of Caki-1 cells overexpressing wild-type MCPIP1 or its D141N mutant, respectively. Gene ontology and KEGG pathway functional analyses showed that these transcripts encoded proteins involved in cell cycle progression, protein folding in the endoplasmic reticulum, hypoxia response and cell signalling. We identified 219 downregulated transcripts in MCPIP1-expressing cells that were either unchanged or upregulated in D141N-expressing cells. We validated downregulation of 15 transcripts belonging to different functional pathways by qRT-PCR. The growth and viability of MCPIP1-expressing cells was reduced because of elevated p21Cip1 levels. MCPIP1-expressing cells also showed reduced levels of DDB1 transcript that encodes component of the E3 ubiquitin ligase that degrades p21Cip1. These results demonstrate that MCPIP1 influences the growth and viability of ccRCC cells by increasing or decreasing the transcript levels for proteins involved in cell cycle progression, protein folding, hypoxia response, and cell signaling.

The FBXW7 tumor suppressor inhibits breast cancer proliferation and promotes apoptosis by targeting MTDH for degradation.

Metadherin (MTDH) is an oncoprotein and is expressed at high levels in a wide variety of human carcinomas, which represents an important genetic determinant and regulates multiple events in tumorigenesis. MTDH promotes breast cancer cell proliferation and tumorigenesis through the activation of numerous signaling pathways. Currently, the mecha- nism regulating MTDH expression is poorly understood. Here we identified that FBXW7, a component of E3 ubiquitin ligase, targets MTDH for ubiquitin-mediated degradation. Forced overexpression of FBXW7 could decrease the level of MTDH protein, and inhibition of endogenous FBXW7 expression remarkably increases the MTDH protein abundance. More importantly, overexpression of FBXW7 could lead to proliferation arrest and apoptosis in breast cancer cells through targeting MTDH degradation. These data suggest that FBXW7, a tumor suppressor, inhibits breast cancer cell prolifera- tion and promotes apoptosis at least partially through targeting MTDH for proteolysis. This new regulatory mechanism of MTDH by FBXW7 represents a new pathway for malignant phenotype turnover in human breast cancer.

FBW7 targets KLF10 for ubiquitin-dependent degradation

FBW7, a key component of SCFFBW7 E3 ubiquitin ligase, targets various proteins for degradation via the conserved Cdc4 phosphodegron (CPD) in substrates. In this study, we report that KLF10 is degraded by FBW7 via a conserved CPD. Through systematic analysis of the degradation of KLF transcription factors by FBW7, we identified KLF10 as a novel degradation target of FBW7. Ectopic expression of FBW7 markedly promoted the degradation of KLF10 while knockdown of endogenous FBW7 increased the protein levels of KLF10. In addition, simultaneous mutations of both threonine 82 (T82) and serine 86 (S86) significantly reduced the FBW7-mediated KLF10 degradation. Moreover, KLF10 containing a conserved putative CPD (TPPXSP) from amino acids 82 to 87, directly interacted with WD40 domain of FBW7 in a phosphorylation-dependent manner. Importantly, FBW7 could reverse the KLF10-mediated inhibition of Smad7 activity. Thus, our study uncovers a novel regulatory mechanism underlying which KLF10 stability and its biological function are mediated by FBW7.

HIV-1 Vpr protein directly loads helicase-like transcription factor (HLTF) onto the CRL4-DCAF1 E3 ubiquitin ligase

The viral protein R (Vpr) is an accessory virulence factor of HIV-1 that facilitates infection in immune cells. Cellular functions of Vpr are tied to its interaction with DCAF1, a substrate receptor component of the CRL4 E3 ubiquitin ligase. Recent proteomic approaches suggested that Vpr degrades helicase-like transcription factor (HLTF) DNA helicase in a proteasome-dependent manner by redirecting the CRL4-DCAF1 E3 ligase. However, the precise molecular mechanism of Vpr-dependent HLTF depletion is not known. Here, using in vitro reconstitution assays, we show that Vpr mediates polyubiquitination of HLTF, by directly loading it onto the C-terminal WD40 domain of DCAF1 in complex with the CRL4 E3 ubiquitin ligase. Mutational analyses suggest that Vpr interacts with DNA-binding residues in the N-terminal HIRAN domain of HLTF in a manner similar to the recruitment of another target, uracil DNA glycosylase (UNG2), to the CRL4-DCAF1 E3 by Vpr. Strikingly, Vpr also engages a second, adjacent region, which connects the HIRAN and ATPase/helicase domains. Thus, our findings reveal that Vpr utilizes common as well as distinctive interfaces to recruit multiple postreplication DNA repair proteins to the CRL4-DCAF1 E3 ligase for ubiquitin-dependent proteasomal degradation.

KLHL7 promotes TUT1 ubiquitination associated with nucleolar integrity: Implications for retinitis pigmentosa

Kelch-like protein 7 (KLHL7) is a component of Cul3-based Cullin-RING ubiquitin ligase. Recent studies have revealed that mutations in klhl7 gene cause several disorders, such as retinitis pigmentosa (RP). Although KLHL7 is considered to be crucial for regulating the protein homeostasis, little is known about its biological functions. In this study, we report that KLHL7 increases terminal uridylyl transferase 1 (TUT1) ubiquitination involved in nucleolar integrity. TUT1 is normally localized in nucleolus; however, expression of KLHL7 facilitates a vulnerability of nucleolar integrity, followed by a decrease of TUT1 localization in nucleolus. On the other hand, pathogenic KLHL7 mutants, which causes an onset of RP, have little effect on both nucleolar integrity and TUT1 localization. Finally, KLHL7 increases TUT1 ubiquitination levels. Taken together, these results imply that KLHL7 is a novel regulator of nucleolus associated with TUT1 ubiquitination. Our study may provide a valuable information to elucidate a pathogenic mechanism of RP.

LncRNA H19 is a major mediator of doxorubicin chemoresistance in breast cancer cells through a cullin4A-MDR1 pathway.

Development of chemoresistance is a persistent problem during cancer treatment. Long non-coding RNAs (LncRNAs) are currently emerging as an integral functional component of the human genome and as critical regulators of cancer development and progression. In the present study, we investigated the role and molecular mechanism of H19 lncRNA in chemoresistance development by using doxorubicin (Dox) resistance in breast cancer cells as a model system. H19 lncRNA expression was significantly increased in anthracycline-treated and Dox-resistant MCF-7 breast cancer cells. This H19 overexpression was contributed to cancer cell resistance to anthracyclines and paclitaxel as knockdown of H19 lncRNA by a specific H19 shRNA in Dox-resistant cells significantly improved the cell sensitivity to anthracyclines and paclitaxel. Furthermore, gene expression profiling analysis revealed that a total of 192 genes were associated with H19-mediated Dox resistance. These genes were enriched in multiple KEGG pathways that are related to chemoresistance. Using genetic and pharmacological approaches, we demonstrated that MDR1 and MRP4 were major effectors of H19-regulated Dox resistance in breast cancer cells as MDR1 and MRP4 expression was markedly elevated in Dox-resistant cells while dramatically reduced when H19 was knocked down. Moreover, we found that CUL4A, an ubiquitin ligase component, was a critical factor bridging H19 lncRNA to MDR1 expression, and a high tumor CUL4A expression was associated with low survival in breast cancer patients treated with chemotherapy. These data suggest that H19 lncRNA plays a leading role in breast cancer chemoresistance, mediated mainly through a H19-CUL4A-ABCB1/MDR1 pathway.

Abstract 5121: PEG(E)-TC11, a novel polyethylene glycol-linked phthalimide derivative, inhibited high-risk MM cell growth in vivo and in vitro via cell cycle G2/M arrest in a CRBN-independent manner

Abstract Multiple myeloma (MM) is one of the hematological malignancy that is characterized by proliferation of malignant plasma cells. Recent advance in the treatment of MM using newly developed drugs, prognosis of the MM patients have been significantly improved. For example, immunomodulatory drugs (IMiDs) such as thalidomide, lenalidomide and pomalidomide have been developed for treatment of MM. However, IMiDs have only limited effects against MM patients with high risk chromosomal abnormalities such as t(4;14) and del17p (high-risk MM). In 2010, it was reported that IMiDs directly bind to cereblon (CRBN), a component of ubiquitin ligase 3 complex, and induced teratogenicity as well as anti-tumor effects. We have previously reported that a novel phthalimide derivative, 2-(2,6-diisopropylphenyl)-5-amino-1H-isoindole-1,3-dione (TC11) induced apoptosis against high-risk MM cells in vivo and in vitro, and inhibited differentiation of osteoclasts. We also reported that TC11 directly bound to α-tubulin and nucleophosmin-1 (NPM1), but did not bind to CRBN. However, TC11 was not well dissolved in water with only 0.02 mg/mL solubility. Therefore, TC11 showed poor absorption into blood and limited anti-tumor activity when it was intraperitoneally administered in tumor-bearing mice. To resolve these problems, we synthesized PEG(E)-TC11, in which TC11 is linked to polyethylene glycol through an ester bond, and consequently enhanced water solubility of PEG(E)-TC11 to 88.9 mg/mL. PEG(E)-TC11 revealed as potent growth inhibitory effect on high-risk MM cells as TC11 in vitro. In pharmacokinetic study, PEG-modification of TC11 improved the peak blood concentration (Cmax) from 2.6 to 24.4 μM and extended elimination half-life (t1/2) from 1.4 to 2.2 hr when 186 μM/kg of these compounds were intraperitoneally injected. More importantly, these pharmacokinetic improvement led to more potent growth inhibition of MM cells in vivo than TC11. We also explored mechanisms of anti-myeloma effect of PEG(E)-TC11 and found that PEG(E)-TC11 induced apoptosis via G2/M cell cycle arrest. However, unlike IMiDs family, BIACORE assay revealed that PEG(E)-TC11 didn’t directly bind to CRBN, indicating that growth inhibitory effect of PEG(E)-TC11 against MM cells was independent of binding to CRBN. In conclusion, PEGylation of TC11 significantly increased water solubility, resulted in potentiated anti-myeloma activity in vivo. Furthermore, PEG(E)-TC11 inhibited cell growth via G2/M arrest in a CRBN-independent manner. Thus, PEG(E)-TC11 is considered as a candidate compound for overcoming high-risk MM. Citation Format: Shuji Aida, Daiju Ichikawa, Kazuki Iida, Masashi Hozumi, Misa Nakamura, Ryo Uozaki, Nahoko Hashimoto, Mikio Okayama, Yuko Yonemura, Noriko Tabata, Taketo Yamada, Maiko Matsushita, Takeshi Sugai, Hiroshi Yanagawa, Yutaka Hattori. PEG(E)-TC11, a novel polyethylene glycol-linked phthalimide derivative, inhibited high-risk MM cell growth in vivo and in vitro via cell cycle G2/M arrest in a CRBN-independent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5121. doi:10.1158/1538-7445.AM2017-5121

Abstract 3908: The mechanism of premetastatic niche formation through FBXW7/NOTCH/CCL2 pathway and clinical trial using CCL2 inhibitor “SK-818” for breast cancer patients

Abstract Background Metastasis is a major cause of death in cancer patients, &amp; elucidation of the mechanisms is expected to provide a basis for the development of new cancer treatment. Premetastatic niche whereby tumors prepare defined organs for metastasized cells is considered to be crucial for the development of metastasis, which consists of host cells such as monocytes, macrophages, &amp; other inflammatory cells. However, the mechanism of how tumor cells communicate with host cells to develop a premetastatic niche remains unclear. FBXW7 is the F-box protein component of an SCF-type ubiquitin ligase, in which it functions as a receptor responsible for substrate recognition followed by the degradation of the substrate. Some part of the substrates of FBXW7 regulates the recruitment of monocytes, macrophages, &amp; other inflammatory cells. Therefore, we hypothesized that expression of FBXW7 in the host cells is a key determinant of the formation of premetastatic niches. Materials &amp; Methods To investigate the role of FBXW7 in the formation of premetastatic niche, we developed bone marrow-specific FBXW7-deficient mice. 1. We transferred B16F10 melanoma cells or E0771 mouse breast cancer cells into the tail vein or mammary fat pad of the mice &amp; the wild type mice to evaluate the metastatic potential to lung. 2. We examined the serum concentrations of various cytokines or chemokines in the mice before &amp; after E0771 cells transplantation using cytokine array. 3. To examine whether the enhanced metastasis apparent in FBXW7-deficient mice is dependent on the CCL2/CCR2 pathway, we treated FBXW7-deficient mice with SK-818, a CCR2 antagonist. 4. We measured the expression of FBXW7 mRNA in peripheral blood of breast cancer patients by qRT-PCR &amp; examined the relationship between FBXW7 mRNA expression &amp; prognosis. Results 1. The extent of lung metastasis of B16F10 or E0771 cell was markedly enhanced in FBXW7-deficient mice. 2. FBXW7-deficient mice exhibited increased serum levels of CCL2. Moreover, accumulation of NOTCH &amp; consequent transcriptional activation of CCL2 was observed in bone marrow-derived stromal cells of FBXW7-deficient mice. 3. Administration of SK-818 blocked the enhancement of lung metastasis in FBXW7-deficient mice. 4. In human breast cancer patients, low FBXW7 mRNA expression in peripheral blood showed high concentration of CCL2 in serum &amp; poor prognosis. Conclusions We found that the FBXW7/NOTCH/CCL2 pathway play a key role in the regulation of cancer metastasis through the formation of premetastatic niches, &amp; that SK-818 inhibit cancer metastasis in mice. SK-818 is currently administered clinically for the treatment of patients with hepatitis B virus infection in Japan. Based on these findings, we have been conducting Phase I clinical trial to assess the safety of SK-818 for breast cancer patients. Citation Format: Miwa Noda, Takaaki Masuda, Naoki Hayashi, Takahiro Kogawa, Shinji Ohno, Keiichi Nakayama, Yohsuke Kuroda, Hidetoshi Eguchi, Shuhei Ito, Masafumi Inomata, Koshi Mimori. The mechanism of premetastatic niche formation through FBXW7/NOTCH/CCL2 pathway and clinical trial using CCL2 inhibitor “SK-818” for breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3908. doi:10.1158/1538-7445.AM2017-3908

MYC Modulation around the CDK2/p27/SKP2 Axis.

MYC is a pleiotropic transcription factor that controls a number of fundamental cellular processes required for the proliferation and survival of normal and malignant cells, including the cell cycle. MYC interacts with several central cell cycle regulators that control the balance between cell cycle progression and temporary or permanent cell cycle arrest (cellular senescence). Among these are the cyclin E/A/cyclin-dependent kinase 2 (CDK2) complexes, the CDK inhibitor p27KIP1 (p27) and the E3 ubiquitin ligase component S-phase kinase-associated protein 2 (SKP2), which control each other by forming a triangular network. MYC is engaged in bidirectional crosstalk with each of these players; while MYC regulates their expression and/or activity, these factors in turn modulate MYC through protein interactions and post-translational modifications including phosphorylation and ubiquitylation, impacting on MYC’s transcriptional output on genes involved in cell cycle progression and senescence. Here we elaborate on these network interactions with MYC and their impact on transcription, cell cycle, replication and stress signaling, and on the role of other players interconnected to this network, such as CDK1, the retinoblastoma protein (pRB), protein phosphatase 2A (PP2A), the F-box proteins FBXW7 and FBXO28, the RAS oncoprotein and the ubiquitin/proteasome system. Finally, we describe how the MYC/CDK2/p27/SKP2 axis impacts on tumor development and discuss possible ways to interfere therapeutically with this system to improve cancer treatment.

F-Box Protein XREP-4 Is a New Regulator of the Oxidative Stress Response in Caenorhabditis elegans.

The transcription factor SKN-1 (Skinhead family member-1) in Caenorhabditis elegans is a homolog of the mammalian Nrf-2 protein and functions to promote oxidative stress resistance and longevity. SKN-1 mediates protection from reactive oxygen species (ROS) via the transcriptional activation of genes involved in antioxidant defense and phase II detoxification. Although many core regulators of SKN-1 have been identified, much remains unknown about this complex signaling pathway. We carried out an ethyl methanesulfonate (EMS) mutagenesis screen and isolated six independent mutants with attenuated SKN-1-dependent gene activation in response to acrylamide. All six were found to contain mutations in F46F11.6/xrep-4 (xenobiotics response pathways-4), which encodes an uncharacterized F-box protein. Loss of xrep-4 inhibits the skn-1-dependent expression of detoxification genes in response to prooxidants and decreases survival of oxidative stress, but does not shorten life span under standard culture conditions. XREP-4 interacts with the ubiquitin ligase component SKR-1 and the SKN-1 principal repressor WDR-23, and knockdown of xrep-4 increases nuclear localization of a WDR-23::GFP fusion protein. Furthermore, a missense mutation in the conserved XREP-4 F-box domain that reduces interaction with SKR-1 but not WDR-23 strongly attenuates SKN-1-dependent gene activation. These results are consistent with XREP-4 influencing the SKN-1 stress response by functioning as a bridge between WDR-23 and the ubiquitin ligase component SKR-1.

A MicroRNA/Ubiquitin Ligase Feedback Loop Regulates Slug-Mediated Invasion in Breast Cancer

The transformation of a normal cell to cancer requires the derail of multiple pathways. Normal signaling in a cell is regulated at multiple stages by the presence of feedback loops, calibration of levels of proteins by their regulated turnover, and posttranscriptional regulation, to name a few. The tumor suppressor protein FBXO31 is a component of the SCF E3 ubiquitin ligase and is required to arrest cells at G1 following genotoxic stresses. Due to its growth-suppression activity, it is underexpressed in many cancers. However, the molecular mechanism underlying the translational regulation of FBXO31 remains unclear. Here we show that the oncogenic microRNAs miR-93 and miR-106a repress FBXO31, resulting in the upregulation of Slug, which is involved in epithelial-mesenchymal transition and cell invasion. FBXO31 targets and ubiquitylates Slug for proteasomal degradation. However, this mechanism is repressed in breast tumors where miR-93 and miR-106a are overexpressed. Our study further unravels an interesting mechanism whereby Slug drives the expression of miR-93 and miR-106a, thus establishing a positive feedback loop to maintain an invasive phenotype. Together, these results establish the presence of interplay between microRNAs and the ubiquitination machinery, which together regulate cancer cell invasion.

HIV-1 Vpu Downmodulates ICAM-1 Expression, Resulting in Decreased Killing of Infected CD4 + T Cells by NK Cells

HIV-1 Vpu is known to alter the expression of numerous cell surface molecules. Given the ever-increasing list of Vpu targets identified to date, we undertook a proteomic screen to discover novel cell membrane proteins modulated by this viral protein. Plasma membrane proteome isolates from Vpu-inducible T cells were subjected to stable isotope labeling of amino acids in cell culture (SILAC)-based mass spectrometry analysis, and putative targets were validated by infection of primary CD4+ T cells. We report here that while intercellular adhesion molecule 1 (ICAM-1) and ICAM-3 are upregulated by HIV-1 infection, expression of Vpu offsets this increase by downregulating these molecules from the cell surface. Specifically, we show that Vpu is sufficient to downregulate and deplete ICAM-1 in a manner requiring the Vpu transmembrane domain and a dual-serine (S52/S56) motif necessary for recruitment of the beta-transducin repeat-containing E3 ubiquitin protein ligase (β-TrCP) component of the Skp, Cullin, F-box (SCFβ-TrCP) E3 ubiquitin ligase. Vpu interacts with ICAM-1 to induce its proteasomal degradation. Interestingly, the E3 ubiquitin ligase component β-TrCP-1 is dispensable for ICAM-1 surface downregulation yet is necessary for ICAM-1 degradation. Functionally, Vpu-mediated ICAM-1 downregulation lowers packaging of this adhesion molecule into virions, resulting in decreased infectivity. Importantly, while Vpu-mediated downregulation of ICAM-3 has a limited effect on the conjugation of NK cells to HIV-1-infected CD4+ T cells, downregulation of ICAM-1 by Vpu results in a reduced ability of NK cells to bind and kill infected T cells. Vpu-mediated ICAM-1 downregulation may therefore represent an evolutionary compromise in viral fitness by impeding the formation of cell-to-cell contacts between immune cells and infected T cells at the cost of decreased virion infectivity.IMPORTANCE The major barrier to eradicating HIV-1 infection is the establishment of treatment-resistant reservoirs early in infection. Vpu-mediated ICAM-1 downregulation may contribute to the evasion of cell-mediated immunity during acute infection to promote viral dissemination and the development of viral reservoirs. By aiding the immune system to clear infection prior to the development of reservoirs, novel treatments designed to disrupt Vpu-mediated ICAM-1 downregulation may be beneficial during acute infection or as a prophylactic treatment.

LIN-23, an E3 Ubiquitin Ligase Component, Is Required for the Repression of CDC-25.2 Activity during Intestinal Development in Caenorhabditis elegans.

Caenorhabditis elegans (C. elegans) utilizes two different cell-cycle modes, binucleations during the L1 larval stage and endoreduplications at four larval moltings, for its postembryonic intestinal development. Previous genetic studies indicated that CDC-25.2 is specifically required for binucleations at the L1 larval stage and is repressed before endoreduplications. Furthermore, LIN-23, the C. elegans β-TrCP ortholog, appears to function as a repressor of CDC-25.2 to prevent excess intestinal divisions. We previously reported that intestinal hyperplasia in lin-23(e1883) mutants was effectively suppressed by the RNAi depletion of cdc-25.2. Nevertheless, LIN-23 targeting CDC-25.2 for ubiquitination as a component of E3 ubiquitin ligase has not yet been tested. In this study, LIN-23 is shown to be the major E3 ubiquitin ligase component, recognizing CDC-25.2 to repress their activities for proper transition of cell-cycle modes during the C. elegans postembryonic intestinal development. In addition, for the first time that LIN-23 physically interacts with both CDC-25.1 and CDC-25.2 and facilitates ubiquitination for timely regulation of their activities during the intestinal development.