USP29 activation mediated by FUBP1 promotes AURKB stability and oncogenic functions in gastric cancer.

BACKGROUND: Gastric cancer is a highly prevalent cancer type and the underlying molecular mechanisms are not fully understood. Ubiquitin-specific peptidase (USP) 29 has been suggested to regulate cell fate in several types of cancer, but its potential role in gastric carcinogenesis remains unclear. METHODS: The expression of USP29 in normal and gastric cancer tissues was analyzed by bioinformatics analysis, immunohistochemistry and immunoblot. Gene overexpression, CRISPR-Cas9 technology, RNAi, and Usp29 knockout mice were used to investigate the roles of USP29 in cell culture, xenograft, and benzo[a]pyrene (BaP)-induced gastric carcinogenesis models. We then delineated the underlying mechanisms using mass spectrometry, co-immunoprecipitation (Co-IP), immunoblot, ubiquitination assay, chromatin immunoprecipitation (ChIP), quantitative real-time PCR (qRT-PCR), and luciferase assays. RESULTS: In this study, we found that USP29 expression was significantly upregulated in gastric cancers and associated with poor patient survival. Ectopic expression of USP29 promoted, while depletion suppressed the tumor growth in vitro and in vivo mouse model. Mechanistically, transcription factor far upstream element binding protein 1 (FUBP1) directly activates USP29 gene transcription, which then interacts with and stabilizes aurora kinase B (AURKB) by suppressing K48-linked polyubiquitination, constituting a FUBP1-USP29-AURKB regulatory axis that medicates the oncogenic role of USP29. Importantly, systemic knockout of Usp29 in mice not only significantly decreased the BaP-induced carcinogenesis but also suppressed the Aurkb level in forestomach tissues. CONCLUSIONS: These findings uncovered a novel FUBP1-USP29-AURKB regulatory axis that may play important roles in gastric carcinogenesis and tumor progression, and suggested that USP29 may become a promising drug target for cancer therapy.

The emerging role of USP29 in cancer and other diseases.

Reversible protein ubiquitination is a key process for maintaining cellular homeostasis. Deubiquitinases, which can cleave ubiquitin from substrate proteins, have been reported to be deeply involved in disease progression ranging from oncology to neurological diseases. The human genome encodes approximately 100 deubiquitinases, most of which are poorly characterized. One of the well-characterized deubiquitases is ubiquitin-specific protease 29 (USP29), which is often upregulated in pathological tissues and plays important roles in the progression of different diseases. Moreover, several studies have shown that deletion of Usp29 in mice does not cause visible growth and developmental defects, indicating that USP29 may be an ideal therapeutic target. In this review, we provide a comprehensive summary of the important roles and regulatory mechanisms of USP29 in cancer and other diseases, which may help us better understand its biological functions and improve future studies to construct suitable USP29-targeted therapy systems.

HILPS, a long noncoding RNA essential for global oxygen sensing in humans.

Adaptation to low levels of oxygen (hypoxia) is a universal biological feature across metazoans. However, the unique mechanisms how different species sense oxygen deprivation remain unresolved. Here, we functionally characterize a novel long noncoding RNA (lncRNA), LOC105369301, which we termed hypoxia-induced lncRNA for polo-like kinase 1 (PLK1) stabilization (HILPS). HILPS exhibits appreciable basal expression exclusively in a wide variety of human normal and cancer cells and is robustly induced by hypoxia-inducible factor 1α (HIF1α). HILPS binds to PLK1 and sequesters it from proteasomal degradation. Stabilized PLK1 directly phosphorylates HIF1α and enhances its stability, constituting a positive feed-forward circuit that reinforces oxygen sensing by HIF1α. HILPS depletion triggers catastrophic adaptation defect during hypoxia in both normal and cancer cells. These findings introduce a mechanism that underlies the HIF1α identity deeply interconnected with PLK1 integrity and identify the HILPS-PLK1-HIF1α pathway as a unique oxygen-sensing axis in the regulation of human physiological and pathogenic processes.

Identification of USP29 as a key regulator of nucleotide biosynthesis in neuroblastoma through integrative analysis of multi-omics data.

Cancer cells show enhanced nucleotide biosynthesis, which is essential for their unlimited proliferation, but the underlying mechanisms are not entirely clear. Ubiquitin specific peptidase 29 (USP29) was reported to sustain neuroblastoma progression by promoting glycolysis and glutamine catabolism; however, its potential role in regulating nucleotide biosynthesis in tumor cells remains unknown. In this study, we depleted endogenous USP29 in MYCN-amplified neuroblastoma SK-N-BE2 cells by sgRNAs and conducted metabolomic analysis in cells with or without USP29 depletion, we found that USP29 deficiency caused a disorder of intermediates involved in glycolysis and nucleotide biosynthesis. De novo nucleotide biosynthesis was analyzed using 13C6 glucose as a tracer under normoxia and hypoxia. The results indicated that USP29-depleted cells showed inhibition of nucleotide anabolic intermediates derived from glucose, and this inhibition was more significant under hypoxic conditions. Analysis of RNA sequencing data in SK-N-BE2 cells demonstrated that USP29 promoted the gene expression of metabolic enzymes involved in nucleotide anabolism, probably by regulating MYC and E2F downstream pathways. These findings indicated that USP29 is a key regulator of nucleotide biosynthesis in tumor cells.

c-MYC-USP49-BAG2 axis promotes proliferation and chemoresistance of colorectal cancer cells in vitro.

Deubiquitinases (DUBs) play critical roles in tumorigenesis and are emerging as potential therapeutic targets. However, it remains less clear which DUBs may play important roles and represent a realistic vulnerability for a particular type of tumor. Here we revealed that Ubiquitin Specific Peptidase 49 (USP49) is transcriptionally activated by c-MYC in colorectal cancer (CRC), and CRC patients with elevated USP49 levels exhibited significantly shorter survival. Knockdown of USP49 markedly inhibited CRC cell proliferation, colony formation, and chemotherapy resistance in vitro. Investigation of mechanisms unravels that USP49 deubiquitinates and stabilizes Bcl-2-Associated Athanogene 2 (BAG2), a well-known protein that antagonizes apoptosis and enables adaptive response of CRC cells. This study identified a novel mechanism by which USP49 promotes CRC cell survival by stabilizing BAG2 through the c-MYC-USP49-BAG2 axis, indicating that USP49 may become a potential therapeutic target for CRC.

The emerging role of deubiquitylating enzymes as therapeutic targets in cancer metabolism.

Cancer cells must rewire cellular metabolism to satisfy the unbridled proliferation, and metabolic reprogramming provides not only the advantage for cancer cell proliferation but also new targets for cancer treatment. However, the plasticity of the metabolic pathways makes them very difficult to target. Deubiquitylating enzymes (DUBs) are proteases that cleave ubiquitin from the substrate proteins and process ubiquitin precursors. While the molecular mechanisms are not fully understood, many DUBs have been shown to be involved in tumorigenesis and progression via controlling the dysregulated cancer metabolism, and consequently recognized as potential drug targets for cancer treatment. In this article, we summarized the significant progress in understanding the key roles of DUBs in cancer cell metabolic rewiring and the opportunities for the application of DUBs inhibitors in cancer treatment, intending to provide potential implications for both research purpose and clinical applications.

USP29 coordinates MYC and HIF1α stabilization to promote tumor metabolism and progression.

Tumor cells must rewire cellular metabolism to satisfy the demands of unbridled growth and proliferation. How these metabolic processes are integrated to fuel cancer cell growth remains largely unknown. Deciphering the regulatory mechanisms is vital to develop targeted strategies for tumor-selective therapies. We herein performed an unbiased and functional siRNA screen against 96 deubiquitinases, which play indispensable roles in cancer and are emerging as therapeutic targets, and identified USP29 as a top candidate essential for metabolic reprogramming that support biosynthesis and survival in tumor cells. Integrated metabolic flux analysis and molecular investigation reveal that USP29 directly deubiquitinates and stabilizes MYC and HIF1α, two master regulators of metabolic reprogramming, enabling adaptive response of tumor cells in both normoxia and hypoxia. Systemic knockout of Usp29 depleted MYC and HIF1α in MYC-driven neuroblastoma and B cell lymphoma, inhibited critical metabolic targets and significantly prolonged survival of tumor-bearing mice. Strikingly, mice homozygous null for the Usp29 gene are viable, fertile, and display no gross phenotypic abnormalities. Altogether, these results demonstrate that USP29 selectively coordinates MYC and HIF1α to integrate metabolic processes critical for cancer cell growth, and therapeutic targeting of USP29, a potentially targetable enzyme, could create a unique vulnerability given deregulation of MYC and HIF1α frequently occurs in human cancers.

Genome-Wide Analysis Identifies Rag1 and Rag2 as Novel Notch1 Transcriptional Targets in Thymocytes.

Recombination activating genes 1 (Rag1) and Rag2 are expressed in immature lymphocytes and essential for generating the vast repertoire of antigen receptors. Yet, the mechanisms governing the transcription of Rag1 and Rag2 remain to be fully determined, particularly in thymocytes. Combining cDNA microarray and ChIP-seq analysis, we identify Rag1 and Rag2 as novel Notch1 transcriptional targets in acute T-cell lymphoblastic leukemia (T-ALL) cells. We further demonstrate that Notch1 transcriptional complexes directly bind the Rag1 and Rag2 locus in not only T-ALL but also primary double negative (DN) T-cell progenitors. Specifically, dimeric Notch1 transcriptional complexes activate Rag1 and Rag2 through a novel cis-element bearing a sequence-paired site (SPS). In T-ALL and DN cells, dimerization-defective Notch1 causes compromised Rag1 and Rag2 expression; conversely, dimerization-competent Notch1 achieves optimal upregulation of both. Collectively, these results reveal Notch1 dimerization-mediated transcription as one of the mechanisms for activating Rag1 and Rag2 expression in both primary and transformed thymocytes. Our data suggest a new role of Notch1 dimerization in compelling efficient TCRß rearrangements in DN progenitors during T-cell development.

Synergistic targeting of CHK1 and mTOR in MYC-driven tumors.

Deregulation of v-myc avian myelocytomatosis viral oncogene homolog (MYC) occurs in a broad range of human cancers and often predicts poor prognosis and resistance to therapy. However, directly targeting oncogenic MYC remains unsuccessful, and indirectly inhibiting MYC emerges as a promising approach. Checkpoint kinase 1 (CHK1) is a protein kinase that coordinates the G2/M cell cycle checkpoint and protects cancer cells from excessive replicative stress. Using c-MYC-mediated T-cell acute lymphoblastic leukemia (T-acute lymphoblastic leukemia) and N-MYC-driven neuroblastoma as model systems, we reveal that both c-MYC and N-MYC directly bind to the CHK1 locus and activate its transcription. CHIR-124, a selective CHK1 inhibitor, impairs cell viability and induces remarkable synergistic lethality with mTOR inhibitor rapamycin in MYC-overexpressing cells. Mechanistically, rapamycin inactivates carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase (CAD), the essential enzyme for the first three steps of de novo pyrimidine synthesis, and deteriorates CHIR-124-induced replicative stress. We further demonstrate that dual treatments impede T-acute lymphoblastic leukemia and neuroblastoma progression in vivo. These results suggest simultaneous targeting of CHK1 and mTOR as a novel and powerful co-treatment modality for MYC-mediated tumors.

Crosstalk between oncogenic MYC and noncoding RNAs in cancer.

The MYC family oncoproteins are deregulated in more than 50 % of human cancers through a variety of mechanisms, such as gene amplification or translocation, super-enhancer activation, aberrant upstream signaling, and altered protein stability. As one of the major drivers in tumorigenesis, MYC regulates the expression of a large number of noncoding genes involved in multiple oncogenic processes. Noncoding RNAs, including miRNA, lncRNA, circRNA, rRNA and tRNA, are also deeply involved in the oncogenic MYC network by functioning as MYC regulators/effectors. In this review, we summarize representative studies depicting the crosstalk between oncogenic MYC and noncoding RNAs in carcinogenesis with the aim of providing potential implications for both basic science and clinical applications.

Regulation of cancer cell metabolism: oncogenic MYC in the driver's seat.

Cancer cells must rewire cellular metabolism to satisfy the demands of unbridled growth and proliferation. As such, most human cancers differ from normal counterpart tissues by a plethora of energetic and metabolic reprogramming. Transcription factors of the MYC family are deregulated in up to 70% of all human cancers through a variety of mechanisms. Oncogenic levels of MYC regulates almost every aspect of cellular metabolism, a recently revisited hallmark of cancer development. Meanwhile, unrestrained growth in response to oncogenic MYC expression creates dependency on MYC-driven metabolic pathways, which in principle provides novel targets for development of effective cancer therapeutics. In the current review, we summarize the significant progress made toward understanding how MYC deregulation fuels metabolic rewiring in malignant transformation.

DUB3 deubiquitinates and stabilizes NRF2 in chemotherapy resistance of colorectal cancer.

The transcription factor nuclear factor (erythroid-derived 2)-like 2 (NRF2) is one of the master regulators that control hundreds of genes containing antioxidant response elements (AREs). The NRF2-ARE pathway plays a complex role in colorectal cancer (CRC). NRF2 activity is known to be regulated by KEAP1-CUL3 E3 ligase-mediated ubiquitination, indicating the importance of deubiquitination regulation. However, the deubiquitinase (DUB) of NRF2 remains unknown. Here, by screening a DUB library, we identified DUB3 as a DUB that remarkably stabilized NRF2. Further experiments demonstrated that DUB3 promoted NRF2 stability and transcriptional activity by decreasing the K48-linked ubiquitination of NRF2. Coimmunoprecipitation studies revealed interactions between NRF2 and DUB3, as well as between KEAP1 and DUB3, indicating that NRF2, DUB3, and KEAP1 formed a large functional complex. Importantly, ectopic expression of DUB3 caused NRF2-dependent chemotherapy resistance in colon cancer cell lines. Thus, to the best of our knowledge, our findings are the first to identify DUB3 as a NRF2 DUB and may provide a new strategy against chemotherapy resistance in CRC and other NRF2-related diseases.

USP49 participates in the DNA damage response by forming a positive feedback loop with p53.

The p53 tumor suppressor is a critical factor in the DNA damage response (DDR), and regulation of p53 stability has a key role in this process. In our study, we identified USP49 as a novel deubiquitinase (DUB) for p53 from a library consisting of 80 DUBs and found that USP49 has a positive effect on p53 transcriptional activity and protein stability. Investigation of the mechanism revealed that USP49 interacts with the N terminus of p53 and suppresses several types of p53 ubiquitination. Furthermore, USP49 rendered HCT116 cells more sensitive to etoposide (Eto)-induced DNA damage and was upregulated in response to several types of cell stress, including DNA damage. Remarkably, USP49 expression was regulated by p53 and USP49 in knockout mice, which are more susceptible to azoxymethane/dextran sulfate sodium (AOM/DSS)-induced colon tumors. These findings suggest that USP49 has an important role in DDR and may act as a potential tumor suppressor by forming a positive feedback loop with p53.

Mycoplasma fermentans deacetylase promotes mammalian cell stress tolerance.

Mycoplasma fermentans is a pathogenic bacterium that infects humans and has potential pathogenic roles in respiratory, genital and rheumatoid diseases. NAD+-dependent deacetylase is involved in a wide range of pathophysiological processes and our studies have demonstrated that expression of mycoplasmal deacetylase in mammalian cells inhibits proliferation but promotes anti-starvation stress tolerance. Furthermore, mycoplasmal deacetylase is involved in cellular anti-oxidation, which correlates with changes in the proapoptotic proteins BIK, p21 and BIM. Mycoplasmal deacetylase binds to and deacetylates the FOXO3 protein, similar with mammalian SIRT2, and affects expression of the FOXO3 target gene BIM, resulting in inhibition of cell proliferation. Mycoplasmal deacetylase also alters the performance of cells under drug stress. This study expands our understanding of the potential molecular and cellular mechanisms of interaction between mycoplasmas and mammalian cells.

Tumor Suppressor PTPRJ Is a Target of miR-155 in Colorectal Cancer.

PTPRJ is known for its antiproliferative role. Loss of heterozygosity (LOH) of PTPRJ has frequently been observed in various human cancers including colorectal cancer (CRC), lung cancer, and breast cancer. However, the function and mechanism of PTPRJ in CRC are not well understood. At the present study, we show that ectopic expression of PTPRJ inhibits cell growth, migration, and invasiveness in CRC cell line HCT116. Moreover, PTPRJ inhibits the tumorigenecity of HCT116 in a xenograft tumor model. MiR-155, the well-known oncomiR in CRC, is identified as an upstream factor of PTPRJ. MiR-155 directly binds to the 3' untranslated region of PTPRJ mRNA and suppresses the mRNA and protein levels of PTPRJ. Furthermore, the growth-promoting and AKT signaling activation effect of miR-155 was abrogated by PTPRJ overexpression, and vice versa. Our study reveals the crucial role of miR-155/PTPRJ/AKT axis in proliferation and migration of CRC cells and suggests a therapeutic potential of PTPRJ. J. Cell. Biochem. 118: 3391-3400, 2017. © 2017 Wiley Periodicals, Inc.

Synthesis and mechanisms of action of novel harmine derivatives as potential antitumor agents.

A series of novel harmine derivatives bearing a benzylindine substituent in position-1 of ß-carboline ring were synthesized and evaluated as antitumor agents. The N2-benzylated ß-carboline derivatives 3a-g represented the most interesting anticancer activities and compound 3c was found to be the most active agent to diverse cancer cell lines such as gastric carcinoma, melanoma and colorectal cancer. Notably, compound 3c showed low toxicity to normal cells. The treatment significantly induced cell apoptosis. Mechanistically, PI3K/AKT signaling pathway mediated compound 3c-induced apoptosis. Compound 3c inhibited phosphorylation of AKT and promoted the production of reactive oxygen species (ROS). The ROS scavenger, LNAC and GSH, could disturb the effect of compound 3c induced apoptosis and PI3K activity inhibitor LY294002 synergistically enhanced compound 3c efficacy. Moreover, the results from nude mice xenograft model showed that compound 3c treatment effectively inhibited tumor growth and decreased tumor weight. Collectively, our results demonstrated that compound 3c exerts apoptotic effect in cancer cells via suppression of phosphorylated AKT and evocation of ROS generation, which suggested that compound 3c might be served as a promising therapeutic agent for cancer treatment.

miR-191 promotes tumorigenesis of human colorectal cancer through targeting C/EBPß.

MicroRNA-191 (miR-191), a small non-coding RNA, is involved in disease development and cancer diagnosis and prognosis. However, how miR-191 functions in colorectal cancer remains largely unclear. In this study, we show that miR-191 is highly expressed in colon tumor tissues, and that inhibition of miR-191 leads to decreased cell growth, proliferation and tumorigenicity in a xenograft model. Overexpression of miR-191 in colorectal cancer cell lines alters cell cycle progression and cell resistance to 5-Fu induced cell apoptosis. Mechanistic studies demonstrated that miR-191 directly binds to the 3'UTR of the C/EBPß mRNA and mediates a decrease in the mRNA and protein expression of C/EBPß. We further showed that C/EBPß induces growth arrest in a colorectal cancer cell line and that its expression is negatively correlated with the miR-191 level in patient samples. Our findings suggest that miR-191 may be a potential gene therapy target for the treatment of colorectal cancer.