UBXN9 inhibits the RNA exosome function to promote T cell control of liver tumorigenesis.

BACKGROUND AND AIMS: Liver tumorigenesis encompasses oncogenic activation and self-adaptation of various biological processes in premalignant hepatocytes to circumvent the pressure of cellular stress and host immune control. Ubiquitin regulatory X domain-containing proteins (UBXNs) participate in the regulation of certain signaling pathways. However, whether UBXN proteins function in the development of liver cancer remains unclear. APPROACH AND RESULTS: Here, we demonstrated that UBXN9 (Alveolar Soft Part Sarcoma Chromosomal Region Candidate Gene 1 Protein/Alveolar Soft Part Sarcoma Locus) expression was decreased in autochthonous oncogene-induced mouse liver tumors and ~47.7% of human HCCs, and associated with poor prognosis in patients with HCC. UBXN9 attenuated liver tumorigenesis induced by different oncogenic factors and tumor growth of transplanted liver tumor cells in immuno-competent mice. Mechanistically, UBXN9 significantly inhibited the function of the RNA exosome, resulting in increased expression of RLR-stimulatory RNAs and activation of the retinoic acid-inducible gene-I-IFN-Ι signaling in tumor cells, and hence potentiated T cell recruitment and immune control of tumor growth. Abrogation of the CD8 + T cell response or inhibition of tumor cell retinoic acid-inducible gene-I signaling efficiently counteracted the UBXN9-mediated suppression of liver tumor growth. CONCLUSIONS: Our results reveal a modality in which UBXN9 promotes the stimulatory RNA-induced retinoic acid-inducible gene-I-interferon signaling that induces anti-tumor T cell response in liver tumorigenesis. Targeted manipulation of the UBXN9-RNA exosome circuit may have the potential to reinstate the immune control of liver tumor growth.

ETV4 potentiates nuclear YAP retention and activities to enhance the progression of hepatocellular carcinoma.

Dysregulation of the Hippo pathway that promotes cell survival, proliferation and tumorigenesis, relays on the coordinated interactions of YAP with the factors that determine YAP translocation and the related transcriptional programming. Here, we demonstrate that ETV4, a transcriptional factor participating in various protumorigenic processes, enhances YAP-mediated transactivation and hepatocellular carcinoma (HCC) progression. Mechanistically, the enhancement of YAP activities is mediated by the interaction between ETV4 and YAP, which not only increases nuclear YAP accumulation but also directly augments the YAP/TEAD4-mediated transcriptional activation in tumor cells. Functionally, the interplay of ETV4 and YAP promotes growth of liver tumor cells, and activates the genes related to myeloid cell recruitment, including CXCL1 and CXCL5, leading to an enriched presence of myeloid-derived suppressive cells and macrophages but a decreased infiltration of T cells and NK cells in transplanted tumors. More importantly, the correlations between YAP activation, the altered immune cell distribution and ETV4 expression are observed in human HCCs. Therefore, our study reveals a functional interaction between ETV4 and YAP that contributes to HCC progression, and provides mechanistic insights into the regulation of nuclear YAP retention and transactivation.

Deubiquitination of the repressor E2F6 by USP22 facilitates AKT activation and tumor growth in hepatocellular carcinoma.

Dysregulated ubiquitination of tumor-related proteins plays a critical role in tumor development and progression. The deubiquitinase USP22 is aberrantly expressed in certain types of cancer and contributes to aggressive tumor progression. However, the precise mechanism underlying the pro-tumorigenic function of USP22 in hepatocellular carcinoma (HCC) remains unclear. Here, we report that E2F6, a pocket protein-independent transcription repressor, is essential for HCC cell growth, and that its activities are controlled by USP22-mediated deubiquitination. USP22 interacts with and stabilizes E2F6, resulting in the transcriptional repression of phosphatase DUSP1. Moreover, the process involving DUSP1 repression by E2F6 strengthens AKT activation in HCC cells. Therefore, these findings provide mechanistic insights into the USP22-mediated control of oncogenic AKT signaling, emphasizing the importance of USP22-E2F6 regulation in HCC development.

USP12 downregulation orchestrates a protumourigenic microenvironment and enhances lung tumour resistance to PD-1 blockade.

Oncogenic activation of KRAS and its surrogates is essential for tumour cell proliferation and survival, as well as for the development of protumourigenic microenvironments. Here, we show that the deubiquitinase USP12 is commonly downregulated in the KrasG12D-driven mouse lung tumour and human non-small cell lung cancer owing to the activation of AKT-mTOR signalling. Downregulation of USP12 promotes lung tumour growth and fosters an immunosuppressive microenvironment with increased macrophage recruitment, hypervascularization, and reduced T cell activation. Mechanistically, USP12 downregulation creates a tumour-promoting secretome resulting from insufficient PPM1B deubiquitination that causes NF-κB hyperactivation in tumour cells. Furthermore, USP12 inhibition desensitizes mouse lung tumour cells to anti-PD-1 immunotherapy. Thus, our findings propose a critical component downstream of the oncogenic signalling pathways in the modulation of tumour-immune cell interactions and tumour response to immune checkpoint blockade therapy.

The deubiquitinase USP16 functions as an oncogenic factor in K-RAS-driven lung tumorigenesis.

K-RAS mutation and molecular alterations of its surrogates function essentially in lung tumorigenesis and malignant progression. However, it remains elusive how tumor-promoting and deleterious events downstream of K-RAS signaling are coordinated in lung tumorigenesis. Here, we show that USP16, a deubiquitinase involved in various biological processes, functions as a promoter for the development of K-RAS-driven lung tumor. Usp16 deletion significantly attenuates K-rasG12D-mutation-induced lung tumorigenesis in mice. USP16 upregulation upon RAS activation averts reactive oxygen species (ROS)-induced p38 activation that would otherwise detrimentally influence the survival and proliferation of tumor cells. In addition, USP16 interacts with and deubiquitinates JAK1, and thereby promoting lung tumor growth by augmenting JAK1 signaling. Therefore, our results reveal that USP16 functions critically in the K-RAS-driven lung tumorigenesis through modulating the strength of p38 and JAK1 signaling.

Fluorinated Carbon Nanotube Superamphiphobic Coating for High-Efficiency and Long-Lasting Underwater Antibiofouling Surfaces.

Biofilm formation on the surface of materials has brought great troubles to various industries. Designing surfaces with long-lasting antibiofouling properties can help restrain primary bacterial and protein attachment and subsequent biofilm formation for a long time, which is also of great significance for industrial applications. In this work, we successfully prepared fluorinated carbon nanotubes through a one-step fluorination method using fluorosilane and fabricated a superamphiphobic coating using a simple spray method. This coating with ultralow surface free energy and stable micro/nano structures achieved highly efficient and long-term underwater antibiofouling properties. Tea, milk, BSA, and bacterial solution can bounce highly on this surface without wetting the surface in air. The long-term existence of the underwater air-bubble layer on the surface of the superamphiphobic coating was observed. Thus, this surface can effectively resist BSA and bacterial attachment (E. coli), and the efficiency, respectively, reaches 97.5 and 98.2%. Even if it is fully soaked in BSA and BS solution for 120 h, the whole surface is still able to repel water, BSA, and BS solution very well. In addition, the coating possessed excellent wear resistance, the CAs of BSA and BS solution just decreased slightly (higher than 158°), and the sliding angles increased slightly (lower than 4°) after 50 tape abrasion cycles. Therefore, this superamphiphobic coating may have promising applications for marine devices, biomedical materials, protective clothing, and chemical shielding.

Targeting POH1 inhibits prostate cancer cell growth and enhances the suppressive efficacy of androgen deprivation and docetaxel.

BACKGROUND: POH1, a member of the JAMM domain containing deubiquitinases, functions in malignant progression of certain types of cancer. However, the role of POH1 in prostate cancer (PCa) remains unclear. METHODS: We performed RNA interference against the JAMM members in PC3 cells and analyzed cell proliferation. POH1 knockdown was established to evaluate the effects of POH1 on cell growth in vitro and in vivo. RNA-sequencing was utilized to explore the molecular details underlying the biological function of POH1 in PCa. The expression of POH1 in PCa tissues was detected by immunohistochemistry. The POH1 inhibitor capzimin was evaluated to explore whether pharmacologically inhibiting POH1 significantly affected PCa cell proliferation alone or enhanced the inhibitory efficacy of docetaxel and androgen deprivation. RESULTS: Functional analyses identified POH1 as a JAMM deubiquitinase that is required for PCa proliferation. Importantly, expression of POH1 was higher in human PCa tissues (PCas) than that in normal prostate tissues, and a positive correlation was detected between elevated POH1 expression and higher pathological grades in PCas. In vivo experiments further demonstrated that depleting POH1 significantly suppressed the growth of PCa cell xenografts. POH1 deficiency profoundly inhibited the expression of a set of genes involving the cell cycle and caused G0/G1 phase arrest. Furthermore, the POH1 inhibitor capzimin phenotypically recapitulated the effects of POH1 knockdown and improved the efficacy of docetaxel and androgen deprivation in PCa cells. CONCLUSIONS: POH1 was overexpressed in PCas and was correlated with pathological grades in human PCas. Inhibiting POH1 by gene silencing or pharmacological inhibition with capzimin suppressed PCa cell growth. Exploring the inhibition of POH1 in combination with other drugs may provide a strategy to benefit patients with PCa.

POH1 contributes to hyperactivation of TGF-ß signaling and facilitates hepatocellular carcinoma metastasis through deubiquitinating TGF-ß receptors and caveolin-1.

BACKGROUND: Hyper-activation of TGF-ß signaling is critically involved in progression of hepatocellular carcinoma (HCC). However, the events that contribute to the dysregulation of TGF-ß pathway in HCC, especially at the post-translational level, are not well understood. METHODS: Associations of deubiquitinase POH1 with TGF-ß signaling activity and the outcomes of HCC patients were examined by data mining of online HCC datasets, immunohistochemistry analyses using human HCC specimens, spearman correlation and survival analyses. The effects of POH1 on the ubiquitination and stability of the TGF-ß receptors (TGFBR1 and TGFBR2) and the activation of downstream effectors were tested by western blotting. Primary mouse liver tissues from polyinosinic:polycytidylic acid (poly I:C)- treated Mx-Cre+, poh1f/f mice and control mice were used to detect the TGF-ß receptors. The metastatic-related capabilities of HCC cells were studied in vitro and in mice. FINDINGS: Here we show that POH1 is a critical regulator of TGF-ß signaling and promotes tumor metastasis. Integrative analyses of HCC subgroups classified with unsupervised transcriptome clustering of the TGF-ß response, metastatic potential and outcomes, reveal that POH1 expression positively correlates with activities of TGF-ß signaling in tumors and with malignant disease progression. Functionally, POH1 intensifies TGF-ß signaling delivery and, as a consequence, promotes HCC cell metastatic properties both in vitro and in vivo. The expression of the TGF-ß receptors was severely downregulated in POH1-deficient mouse hepatocytes. Mechanistically, POH1 deubiquitinates the TGF-ß receptors and CAV1, therefore negatively regulates lysosome pathway-mediated turnover of TGF-ß receptors. CONCLUSION: Our study highlights the pathological significance of aberrantly expressed POH1 in TGF-ß signaling hyperactivation and aggressive progression in HCC.

Requirement for POH1 in differentiation and maintenance of regulatory T cells.

Foxp3-expressing regulatory T (Treg) cells are essential for averting autoimmune diseases and maintaining immune homeostasis. However, the molecular mechanisms underlying the development and maintenance of Treg cells are still unclear. Here, we found that T cell-specific deletion of the gene encoding the deubiquitinase POH1 compromised the development of mature T cells, especially CD4+Foxp3+ Treg cells. Moreover, POH1 deficiency significantly attenuated the transition of CD25+ Treg cell precursors into Foxp3+ Treg cells accompanied by downregulation of interleukin 2 (IL-2)-STAT5 signaling. Deletion of POH1 in generated CD4+Foxp3+ Treg cells led to an early onset of fetal autoimmune disorders and a decrease in the pool size of peripheral Treg cells in mice, which were mostly due to decreased expansion of these cells. Thus, these results revealed that POH1 has a pivotal role in the development and maintenance of CD4+Foxp3+ Treg cells and contributes to immune tolerance.

Dynein axonemal intermediate chain 2 plays a role in gametogenesis by activation of Stat3.

We previously identified the mouse dynein axonemal intermediate chain 2 (Dnaic2) gene. This gene expresses a component of the axonemal dynein complex that functions in cilia or flagella. We found that overexpression of Dnaic2 results in female subfertility and male infertility. In this study, we generated Dnaic2 knockdown (KD) mice and identified the potential regulatory mechanisms involved in Dnaic2 function. For phenotype analysis, we found that body weight was lighter and size was smaller in Dnaic2 KD mice than in wild-type mice. A total of 45% of these Dnaic2 KD mice were infertile due to sperm abnormalities in males, or had oocyte abnormalities and pathological changes in the tunica mucosa in the oviduct of females. Moreover, Dnaic2 overexpression enhanced the expression of proliferating cell nuclear antigen (PCNA) in the ovaries, which suggested that Dnaic2 stimulated proliferation of cells in the ovaries. However, PCNA expression in the testis of Dnaic2-overexpressed mice was lower than that in controls. Additionally, the ratio of Bax/B-cell lymphoma-2(Bcl-2) in the testis of Dnaic2-overexpressed mice was higher than that in controls, which suggested that Dnaic2 inhibited cellular proliferation in the testis. To examine the molecular action of Dnaic2, immunoprecipitation analysis was used and showed that Dnaic2 protein interacted with signal transducer and activator of transcription 3 (Stat3). Molecular modelling analysis showed that Dnaic2 bound with the linker and SH2 domains of Stat3. Furthermore, overexpression of Dnaic2 promoted phosphorylation of Stat3. In conclusion, our study suggests that Dnaic2 plays a role in oogenesis and spermatogenesis by activation of Stat3.

USP1 inhibition destabilizes KPNA2 and suppresses breast cancer metastasis.

Metastatic progression is the main cause of mortality in breast cancer, necessitating the determination of the molecular events driving this process for the development of new therapeutic approaches. Here, we demonstrate that hyperactivation of the deubiquitinase USP1 contributes to breast cancer metastasis. Upregulated USP1 expression in primary breast cancer specimens correlates with metastatic progression and poor prognosis in breast cancer patients. USP1 enhances the expression of a number of pro-metastatic genes in breast cancer cells, promotes cell migration and invasion in vitro, and facilitates lung metastasis of breast cancer cells. Moreover, USP1-mediated deubiquitination and stabilization of KPNA2 are revealed as the downstream events crucial for USP1-pro-metastatic function. Most importantly, pharmacological intervention of USP1 function by pimozide or ML323 significantly represses breast cancer metastasis in mice, suggesting a rationale for using USP1 inhibitors for treatment of patients with breast cancer. Taken together, our results establish USP1 as a promoter of breast cancer metastasis and provide evidence for the potential practice of USP1 targeting in the treatment of breast cancer.

POH1 deubiquitinates pro-interleukin-1ß and restricts inflammasome activity.

Inflammasome activation is essential for host defence against invading pathogens, but is also involved in various forms of inflammatory diseases. The processes that control inflammasome activity are thus important for averting excessive immune responses and tissue damage. Here we show that the deubiquitinase POH1 negatively regulates the immune response triggered by inflammasome activation. POH1 deficiency in macrophages enhances mature IL-1ß production without significant alterations in inflammasome priming and ASC-caspase-1 activation. In WT macrophages, POH1 interacts with and deubiquitinates pro-IL-1ß by decreasing the K63-linked polyubiquitin chains, as well as decreases the efficacy of pro-IL-1ß cleavage. Furthermore, myeloid cell-specific deletion of POH1 aggravates lipopolysaccharide-induced systemic inflammation and alum-induced peritonitis inflammatory responses in vivo. Our study thereby reveals that POH1-mediated deubiquitination of pro-IL-1ß is an important regulatory event that restrains inflammatory responses for the maintenance of immune homeostasis.

USP16 Downregulation by Carboxyl-terminal Truncated HBx Promotes the Growth of Hepatocellular Carcinoma Cells.

Hepatitis B virus (HBV) infection is a major factor that contributes to the development of hepatocellular carcinoma (HCC). HBV X protein (HBx) has been shown to accelerate HCC progression by promoting tumour growth and metastasis. In the clinic, carboxyl-terminal truncated HBx (Ct-HBx) proteins are frequently present in HCC tumour tissues, but not in non-tumorous tissues. In this study, we analysed deubiquitinase expression profiles in cells with or without ectopic expression of the Ct-HBx proteins and observed that the expression of ubiquitin specific peptidase 16 (USP16) was substantially inhibited by Ct-HBx proteins. Liver tumour cells with forced down-regulation of USP16 exhibited increased capabilities for colony formation and tumour growth in vivo. In addition, USP16 inhibition promoted stem-like properties in tumour cells, as evidenced by their spheroid formation and chemo-responsiveness. Furthermore, ectopic expression of USP16 in tumour cells significantly abrogated the tumour promoting activities of the Ct-HBx proteins (HBxΔ35), leading to decreased tumour cell viability and tumour growth. In human HCCs, USP16 was frequently downregulated, and the decreased expression of USP16 was correlated with high tumour stages and poor differentiation status. Taken together, our study suggests that USP16 downregulation is a critical event in Ct-HBx-mediated promotion of HCC tumorigenicity and malignancy.

POH1 deubiquitylates and stabilizes E2F1 to promote tumour formation.

Hyperactivation of the transcriptional factor E2F1 occurs frequently in human cancers and contributes to malignant progression. E2F1 activity is regulated by proteolysis mediated by the ubiquitin-proteasome system. However, the deubiquitylase that controls E2F1 ubiquitylation and stability remains undefined. Here we demonstrate that the deubiquitylase POH1 stabilizes E2F1 protein through binding to and deubiquitylating E2F1. Conditional knockout of Poh1 alleles results in reduced E2F1 expression in primary mouse liver cells. The POH1-mediated regulation of E2F1 expression strengthens E2F1-downstream prosurvival signals, including upregulation of Survivin and FOXM1 protein levels, and efficiently facilitates tumour growth of liver cancer cells in nude mice. Importantly, human hepatocellular carcinomas (HCCs) recapitulate POH1 regulation of E2F1 expression, as nuclear abundance of POH1 is increased in HCCs and correlates with E2F1 overexpression and tumour growth. Thus, our study suggests that the hyperactivated POH1-E2F1 regulation may contribute to the development of liver cancer.

SIP1 is a downstream effector of GADD45G in senescence induction and growth inhibition of liver tumor cells.

Cellular senescence evasion caused by the inactivation of tumor suppressive programs is implicated in tumor initiation and therapeutic resistance. Our previous study has shown that the downregulation of growth arrest and DNA damage 45G (GADD45G) contributes to senescence bypass in hepatocellular carcinoma (HCC). Here, we report that the Smad-interacting protein-1 (SIP1) is transcriptionally activated and functions critically in the GADD45G-induced tumor cell senescence. Knockdown of SIP1 significantly abrogates the suppressive effects of GADD45G on the growth of xenografted liver tumor in vivo. The essential role of SIP1 in GADD45G activities is further validated in the model of the proteasome inhibitor MG132-induced cell senescence. We further show that JNK but not p38 MAPK activation is involved in the GADD45G-mediated SIP1 upregulation, and that JNK inhibition counteracts the GADD45G-induced cellular senescence. More importantly, we show that GADD45G and SIP1 expression are coincidently downregulated in primary human HCC tissues. Together, our results establish that the dowregulation of GADD45G-SIP1 axis may contribute to cellular senescence evasion and HCC development.

Transcription factor KLF9 suppresses the growth of hepatocellular carcinoma cells in vivo and positively regulates p53 expression.

Krüppel-like factor 9 (KLF9) is known to be a tumor suppressor gene in colorectal tumors and glioblastoma; however, the functional status and significance of KLF9 in hepatocellular carcinoma (HCC) is unclear. We report here that KLF9 is downregulated in HCC tissues. Restoration of KLF9 significantly inhibited growth and caused apoptosis in SK-Hep1 and HepG2 cells. We found that KLF9 positively regulated p53 levels by directly binding to GC boxes within the proximal region of the p53 promoter. Moreover, in the presence of cycloheximide, KLF9 significantly increased p53 stability in HCC cells. Remarkably, ectopic expression of KLF9 was sufficient to delay the onset of tumors and to promote regression of the established tumors in vivo, suggesting that KLF9 plays a critical role in HCC development and that pharmacological or genetic activation of KLF9 may have potential in the treatment of HCC.

GADD45 proteins: roles in cellular senescence and tumor development.

The growth arrest and DNA damage 45 (GADD45) family genes regulate DNA repair, cell cycle, cell survival, apoptosis, senescence, and DNA demethylation in the cells under various stress stimuli, such as oxidative stress, UV radiation, and oncogenic stress. Recent studies have provided important insights regarding how different oncogenic stresses activate GADD45 signaling pathway and lead to disparate influences on tumor initiation. In this review, we discuss the deregulation and cellular function of GADD45 proteins in the context of cancer development. We also highlight recent advances in exploring the tumor suppressive function of GADD45 proteins-triggered cellular senescence.

The combinatory effects of PPAR-γ agonist and survivin inhibition on the cancer stem-like phenotype and cell proliferation in bladder cancer cells.

Strategies for peroxisome proliferator-activated receptor (PPAR) activation or survivin inhibition have potential for cancer therapy. However, whether the combination of these two approaches can be developed as a rational regimen with enhanced efficiency in the inhibition of tumor cells remains to be determined. In this study, the combinatory effect of PPAR-γ agonist and survivin inhibition on bladder cancer cells was investigated. T24 and 5637 cells were treated with 15d-PGJ(2) to determine whether 15d-PGJ(2) had an inhibitory effect. Cell viability and proliferation were analyzed and efficiency of survivin siRNAs was assessed using western blot analysis. The results showed that, in the human bladder cancer cell lines T24 and 5637, the natural PPAR-γ ligand 15d-PGJ(2) significantly decreased cell proliferation and loci formation. The increase in the proportion of apoptotic cells was observed in the cells 48 h after 15d-PGJ(2) treatment. Furthermore, 15d-PGJ(2) substantially inhibited the levels of stemness-related genes in these cells. The ability of sphere formation was markedly suppressed in the cells treated with 15d-PGJ(2). More importantly, the downregulation of survivin with siRNAs significantly enhanced the 15d-PGJ(2)-mediated induction of cell apoptosis and inhibition of sphere formation. Accordingly, we also found that survivin inhibition significantly enhanced 15d-PGJ(2)-induced production of reactive oxygen species (ROS) in bladder cancer cells. Taken together, these findings suggest that the combination of 15d-PGJ(2) and survivin inhibition play a potentially role in the therapeutical manipulation of bladder cancer.

KLF9, a transcription factor induced in flutamide-caused cell apoptosis, inhibits AKT activation and suppresses tumor growth of prostate cancer cells.

BACKGROUND: Kruppel-like factors (KLFs) are involved in various biological processes; emerging studies have indicated that KLF9 plays a critical role in regulating tumorigenesis. The role of KLF9 in prostate cancer (PCa), however, has not yet been investigated. METHODS: The expression of KLF members, AKT- and apoptosis-related proteins were analyzed by Western blot or qRT-PCR. Tet-On inducible KLF9 expression was established for the evaluation of the effects of KLF9 on cell proliferation, apoptosis, and xenograft tumor growth in nude mice. Cell cycle and apoptosis were determined by flow cytometry. RESULTS: KLF9 was induced in a time-dependent manner in flutamide-caused apoptosis, and knockdown of KLF9 significantly decreased flutamide-induced growth inhibition and apoptosis in LNCaP cells. The levels of KLF9 were relatively lower in PCa cell lines, particularly in androgen-independent cell lines compared with those in nontumorous prostate epithelial cell lines. Overexpression of KLF9 dramatically suppressed cell proliferation and caused cell cycle arrest in the G2/M phase and cell apoptosis in the androgen-independent cell lines, PC3 and DU145. Intriguingly, KLF9 expression severely suppressed the activation of AKT and its downstream targets. AKT reactivation partially rescued the KLF9-mediated inhibitory effects on the proliferation of PCa cells. More importantly, we found that KLF9 overexpression efficiently inhibited the xenograft tumor growth of PCa cells. CONCLUSIONS: These data collectively showing that KLF9 substantially inhibits AKT activation and abrogates tumor growth of PCa cells, suggest the potential of either genetic or pharmacological activation of KLF9 in the therapeutic treatment of castration-resistant PCa.