Search

Lysyl oxidase-like 2 (LOXL2) is a member of the scavenger receptor cysteine-rich (SRCR) repeat carrying LOX family. Although LOXL2 is suspected to be involved in histone association and chromatin modification, the role of LOXL2 in epigenetic regulation during tumorigenesis and cancer progression remains unclear. Here, we report that nuclear LOXL2 associates with histone H3 and catalyzes H3K36ac deacetylation and deacetylimination. Both the N-terminal SRCR repeats and the C-terminal catalytic domain of LOXL2 carry redundant deacetylase catalytic activity. Overexpression of LOXL2 markedly reduced H3K36 acetylation and blocked H3K36ac-dependent transcription of genes, including c-MYC, CCND1, HIF1A, and CD44. Consequently, LOXL2 overexpression reduced cancer cell proliferation in vitro and inhibited xenograft tumor growth in vivo. In contrast, LOXL2 deficiency resulted in increased H3K36 acetylation and aberrant expression of H3K36ac-dependent genes involved in multiple oncogenic signaling pathways. Female LOXL2-deficient mice spontaneously developed uterine hypertrophy and uterine carcinoma. Moreover, silencing LOXL2 in cancer cells enhanced tumor progression and reduced the efficacy of cisplatin and anti-programmed cell death 1 (PD-1) combination therapy. Clinically, low nuclear LOXL2 expression and high H3K36ac levels corresponded to poor prognosis in uterine endometrial carcinoma patients. These results suggest that nuclear LOXL2 restricts cancer development in the female reproductive system via the regulation of H3K36ac deacetylation. Significance: LOXL2 loss reprograms the epigenetic landscape to promote uterine cancer initiation and progression and repress the efficacy of anti–PD-1 immunotherapy, indicating that LOXL2 is a tumor suppressor.

Supplementary table

Supplementary table

Correlation assays of expression TRIM59 with SOX9 and EGFR with SOX9

Figure S.4

Supplementary Figure

Lysyl oxidase-like 2 (LOXL2) is a member of the scavenger receptor cysteine-rich (SRCR) repeat carrying LOX family. Although LOXL2 is suspected to be involved in histone association and chromatin modification, the role of LOXL2 in epigenetic regulation during tumorigenesis and cancer progression remains unclear. Here, we report that nuclear LOXL2 associates with histone H3 and catalyzes H3K36ac deacetylation and deacetylimination. Both the N-terminal SRCR repeats and the C-terminal catalytic domain of LOXL2 carry redundant deacetylase catalytic activity. Overexpression of LOXL2 markedly reduced H3K36 acetylation and blocked H3K36ac-dependent transcription of genes, including c-MYC, CCND1, HIF1A, and CD44. Consequently, LOXL2 overexpression reduced cancer cell proliferation in vitro and inhibited xenograft tumor growth in vivo. In contrast, LOXL2 deficiency resulted in increased H3K36 acetylation and aberrant expression of H3K36ac-dependent genes involved in multiple oncogenic signaling pathways. Female LOXL2-deficient mice spontaneously developed uterine hypertrophy and uterine carcinoma. Moreover, silencing LOXL2 in cancer cells enhanced tumor progression and reduced the efficacy of cisplatin and anti-programmed cell death 1 (PD-1) combination therapy. Clinically, low nuclear LOXL2 expression and high H3K36ac levels corresponded to poor prognosis in uterine endometrial carcinoma patients. These results suggest that nuclear LOXL2 restricts cancer development in the female reproductive system via the regulation of H3K36ac deacetylation.Significance:LOXL2 loss reprograms the epigenetic landscape to promote uterine cancer initiation and progression and repress the efficacy of anti–PD-1 immunotherapy, indicating that LOXL2 is a tumor suppressor.

Supplementary table

Primers for qPCR

Purpose:Metallothionein 2A (MT2A) suppresses the progression of human gastric cancer potentially through an “MT2A–NF-κB pathway” with unclear mechanisms. This study explored the role of a transcription factor, myeloid zinc-finger 1 (MZF1), in MT2A-NF-κB pathway and its clinical significance in gastric cancer.Experimental Design:MZF1 expression and function in gastric cancer were investigated in vitro and in vivo. The relationship between MZF1 and MT2A was determined by gain-of-function and loss-of-function assays in gastric cancer cells and an immortalized gastric cell line GES-1. The prognostic value of MZF1 expression in association with MT2A was evaluated using IHC in two cohorts.Results:MZF1 was epigenetically silenced in human gastric cancer cell lines and primary tumors. Overexpression of MZF1 in gastric cancer cells suppressed cell proliferation and migration, as well as the growth of xenograft tumors in nude mice. Knocking-down of MZF1 transformed GES-1 cells into a malignant phenotype characterized by increased cell growth and migration. Mechanistically, MZF1 was upregulated in both GC and GES-1 cells by MT2A ectopically expressed or induced upon treatment with a garlic-derived compound, diallyl trisulfide (DATS). MZF1 associated with MT2A was colocalized in the nuclei of GES-1 cells to target the promoter of NF-κB inhibitor alpha (NFKBIA). Clinically, MT2A and MZF1 were progressively downregulated in clinical specimens undergoing gastric malignant transformation. Downregulation of MT2A and MZF1 was significantly correlated with poorer patient prognosis.Conclusions:MT2A exerts its anti-gastric cancer effects by complexing with MZF1 to target NFKBIA. MT2A/MZF1 may serve as a valuable prognostic marker and a novel therapeutic target for human gastric cancer.

Supplementary Materials and Methods

Supplementary table

Supplementary table

Supplementary table

Supplementary table

Supplementary table

Supplementary information

Supplementary table

Aberrant EGFR signaling is a common driver of glioblastoma (GBM) pathogenesis; however, the downstream effectors that sustain this oncogenic pathway remain unclarified. Here we demonstrate that tripartite motif-containing protein 59 (TRIM59) acts as a new downstream effector of EGFR signaling by regulating STAT3 activation in GBM. EGFR signaling led to TRIM59 upregulation through SOX9 and enhanced the interaction between TRIM59 and nuclear STAT3, which prevents STAT3 dephosphorylation by the nuclear form of T-cell protein tyrosine phosphatase (TC45), thereby maintaining transcriptional activation and promoting tumorigenesis. Silencing TRIM59 suppresses cell proliferation, migration, and orthotopic xenograft brain tumor formation of GBM cells and glioma stem cells. Evaluation of GBM patient samples revealed an association between EGFR activation, TRIM59 expression, STAT3 phosphorylation, and poor prognoses. Our study identifies TRIM59 as a new regulator of oncogenic EGFR/STAT3 signaling and as a potential therapeutic target for GBM patients with EGFR activation.Significance: These findings identify a novel component of the EGFR/STAT3 signaling axis in the regulation of glioma tumorigenesis. Cancer Res; 78(7); 1792–804. ©2018 AACR.

Riboflavin is an essential micronutrient for normal cellular growth and function. Lack of dietary riboflavin is associated with an increased risk for esophageal squamous cell carcinoma (ESCC). Previous studies have identified that the human riboflavin transporter SLC52A3a isoform (encoded by SLC52A3) plays a prominent role in esophageal cancer cell riboflavin transportation. Furthermore, SLC52A3 gene single nucleotide polymorphisms rs3746804 (T>C, L267P) and rs3746803 (C >T, T278M) are associated with ESCC risk. However, whether SLC52A3a (p.L267P) and (p.T278M) act in riboflavin transportation in esophageal cancer cell remains inconclusive. Here, we constructed the full-length SLC52A3a protein fused to green fluorescent protein (GFP-SLC52A3a-WT and mutants L267P, T278M, and L267P/T278M). It was confirmed by immunofluorescence-based confocal microscopy that SLC52A3a-WT, L267P, T278M, and L267P/T278M expressed in cell membrane, as well as in a variety of intracellular punctate structures. The live cell confocal imaging showed that SLC52A3a-L267P and L267P/T278M increased the intracellular trafficking of SLC52A3a in ESCC cells. Fluorescence recovery after photobleaching of GFP-tagged SLC52A3a meant that intracellular trafficking of SLC52A3a-L267P and L267P/T278M was rapid dynamics process, leading to its stronger ability to transport riboflavin. Taken together, the above results indicated that the rs3746804 (p.L267P) polymorphism promoted intracellular trafficking of SLC52A3a and riboflavin transportation in ESCC cells.

Histone deacetylases 1 (HDAC1), an enzyme that functions to remove acetyl molecules from ε-NH3 groups of lysine in histones, eliminates the histone acetylation at the promoter regions of tumor suppressor genes to block their expression during tumorigenesis. However, it remains unclear why HDAC1 fails to impair oncogene expression. Here we report that HDAC1 is unable to occupy at the promoters of oncogenes but maintains its occupancy with the tumor suppressors due to its interaction with CREPT (cell cycle-related and expression-elevated protein in tumor, also named RPRD1B), an oncoprotein highly expressed in tumors. We observed that CREPT competed with HDAC1 for binding to oncogene (such as CCND1, CLDN1, VEGFA, PPARD and BMP4) promoters but not the tumor suppressor gene (such as p21 and p27) promoters by a chromatin immunoprecipitation (ChIP) qPCR experiment. Using immunoprecipitation experiments, we deciphered that CREPT specifically occupied at the oncogene promoter via TCF4, a transcription factor activated by Wnt signaling. In addition, we performed a real-time quantitative PCR (qRT-PCR) analysis on cells that stably over-expressed CREPT and/or HDAC1, and we propose that HDAC1 inhibits CREPT to activate oncogene expression under Wnt signaling activation. Our findings revealed that HDAC1 functions differentially on tumor suppressors and oncogenes due to its interaction with the oncoprotein CREPT.

Background Signal transducer and activator of transcription 3 (STAT3) has been shown to upregulate gene transcription during tumorigenesis. However, how STAT3 initiates transcription remains to be exploited. This study is to reveal the role of CREPT (cell cycle-related and elevated-expression protein in tumours, or RPRD1B) in promoting STAT3 transcriptional activity. Methods BALB/c nude mice, CREPT overexpression or deletion cells were employed for the assay of tumour formation, chromatin immunoprecipitation, assay for transposase-accessible chromatin using sequencing. Results We demonstrate that CREPT, a recently identified oncoprotein, enhances STAT3 transcriptional activity to promote tumorigenesis. CREPT expression is positively correlated with activation of STAT3 signalling in tumours. Deletion of CREPT led to a decrease, but overexpression of CREPT resulted in an increase, in STAT3-initiated tumour cell proliferation, colony formation and tumour growth. Mechanistically, CREPT interacts with phosphorylated STAT3 (p-STAT3) and facilitates p-STAT3 to recruit p300 to occupy at the promoters of STAT3-targeted genes. Therefore, CREPT and STAT3 coordinately facilitate p300-mediated acetylation of histone 3 (H3K18ac and H3K27ac), further augmenting RNA polymerase II recruitment. Accordingly, depletion of p300 abolished CREPT-enhanced STAT3 transcriptional activity. Conclusions We propose that CREPT is a co-activator of STAT3 for recruiting p300. Our study provides an alternative strategy for the therapy of cancers related to STAT3.

Exosomes released by mesenchymal stem cells (MSCs) are thought to function as extensions of the MSCs. However, it remains unclear whether exosomes derived from human umbilical cord MSCs (HUMSCs) possess immunoregulatory functions in rheumatoid arthritis. We report that when mice with collagen-induced arthritis were injected with exosomes derived from HUMSC (HUMSC-Exo), their paws became less swollen, and they had lower serum pro-inflammatory cytokine and anti-collagen IgG levels, and decreased synovial hyperplasia. The HUMSC-Exo appeared to restore the balance between Th17 and Treg cells, and this effect was accompanied by reduced IL-17 and enhanced TGF-β and IL-10 levels. These findings suggest that HUMSC-Exo function as important regulator of the balance between Th1/Th17 and Treg cells during immune and inflammatory responses.

Cancers remain a threat to human health due to the lack of effective therapeutic strategies. Great effort has been devoted to the discovery of drug targets to treat cancers, but novel oncoproteins still need to be unveiled for efficient therapy. Methods: We show that CREPT is highly expressed in pancreatic cancer and is associated with poor disease-free survival. CREPT overexpression promotes but CREPT deletion blocks colony formation and proliferation of pancreatic cancer cells. To provide a proof of concept for CREPT as a new target for the inhibition of pancreatic cancer, we designed a cell-permeable peptide-based proteolysis targeting chimera (PROTAC), named PRTC, based on the homodimerized leucine-zipper-like motif in the C-terminus domain of CREPT to induce its degradation in vivo. Results: PRTC has high affinity for CREPT, with Kd = 0.34 +/- 0.11 μM and is able to permeate into cells because of the attached membrane-transportable peptide RRRRK. PRTC effectively induces CREPT degradation in a proteasome-dependent manner. Intriguingly, PRTC inhibits colony formation, cell proliferation, and motility in pancreatic cancer cells and ultimately impairs xenograft tumor growth, comparable to the effect of CREPT deletion. Conclusions: PRTC-induced degradation of CREPT leads to inhibition of tumor growth, which is promising for the development of new drugs against pancreatic cancer. In addition, using an interacting motif based on the dimerized structure of proteins may be a new way to design a PROTAC aiming at degrading any protein without known interacting small molecules or peptides.

Understanding the potential mechanisms and processes of leaf photosynthesis in response to elevated CO

Cathepsins play a role in regulation of cell function through their presence in the cell nucleus. However, the role of Cathepsin K (Ctsk) as an epigenetic regulator in osteoclasts remains unknown. Our data demonstrated that

Oxidative DNA damage contributes to aging and the pathogenesis of numerous human diseases including cancer. 8-hydroxyguanine (8-oxoG) is the major product of oxidative DNA lesions. Although OGG1-mediated base excision repair is the primary mechanism for 8-oxoG removal, DNA mismatch repair has also been implicated in processing oxidative DNA damage. However, the mechanism of the latter is not fully understood. Here, we treated human cells defective in various 8-oxoG repair factors with H

T helper cells, especially Th1 and Th17 cells, were reported to play a pivotal role in the pathogenesis of inflammatory bowel disease (IBD). However, the underlying factors regulating T cell functions in IBD progression remain to be fully elucidated. Here, we revealed that IL-17RD/Sef exacerbates DSS-induced colitis by regulating the balance of T cell subsets and their secretion of associated cytokines. We also observed that IL-17RD/Sef promotes colitis-associated tumorigenesis and negatively correlates with survival in both mouse and colorectal cancer patients. Our results suggested that IL-17RD/Sef functions as a regulator of T cell subsets to promote the inflammatory responses in the pathogenesis of IBD and colitis-associated colon cancer.

T helper 1 (Th1) immunity is typically viewed as a critical adaptation by vertebrates against intracellular pathogens. Identifying novel targets to enhance Th1 cell differentiation and function is increasingly important for anti-infection immunity. Here, through small-molecule screening focusing on epigenetic modifiers during the in vitro Th1 cell differentiation process, we identified that the selective histone deacetylase 6 (HDAC6) inhibitors ricolinostat and nexturastat A (Nex A) promoted Th1 cell differentiation. HDAC6-depleted mice exhibit elevation of Th1 cell differentiation, and decreased severity of Listeria monocytogenes infection. Mechanistically, HDAC6 directly deacetylated CBP-catalyzed acetylation of signal transducer and activator of transcription 4 (STAT4)-lysine (K) 667 via its enzymatic activity. Acetylation of STAT4-K667 is required for JAK2-mediated phosphorylation and activation of STAT4. Stat4supK667R/supmutant mice lost the ability to normally differentiate into Th1 cells and developed severe Listeria infection. Our study identifies acetylation of STAT4-K667 as an essential signaling event for Th1 cell differentiation and defense against intracellular pathogen infections, and highlights the therapeutic potential of HDAC6 inhibitors for controlling intracellular pathogen infections.

Summary The mammalian target of rapamycin (mTOR) is a serine-threonine kinase involved in cellular innate immunity, metabolism, and senescence. FK506-binding protein 12 (FKBP12) inhibits mTOR kinase activity via direct association. The FKBP12-mTOR association can be strengthened by the immunosuppressant rapamycin, but the underlying mechanism remains elusive. We show here that the FKBP12-mTOR association is tightly regulated by an acetylation–deacetylation cycle. FKBP12 is acetylated on the lysine cluster (K45/K48/K53) by CREB-binding protein (CBP) in mammalian cells in response to nutrient treatment. Acetyl-FKBP12 associates with CBP acetylated Rheb. Rapamycin recruits SIRT2 with a high affinity for FKBP12 association and deacetylation. SIRT2-deacetylated FKBP12 then switches its association from Rheb to mTOR. Nutrient-activated mTOR phosphorylates IRF3S386 for the antiviral response. In contrast, rapamycin strengthening FKBP12-mTOR association blocks mTOR antiviral activity by recruiting SIRT2 to deacetylate FKBP12. Hence, on/off mTOR activity in response to environmental nutrients relies on FKBP12 acetylation and deacetylation status in mammalian cells., Graphical abstract, Highlights • FKBP12-mTOR association is tightly regulated by an acetylation–deacetylation cycle • SIRT2 is responsible for FKBP12 deacetylation • Acetylation of Rheb is indispensable to mTOR activation • mTOR phosphorylates IRF3 S386 for type-I interferon gene expression, Biochemistry; Protein; Molecular biology

Background Fascin is crucial for cancer cell filopodium formation and tumor metastasis, and is functionally regulated by post‐translational modifications. However, whether and how Fascin is regulated by acetylation remains unclear. This study explored the regulation of Fascin acetylation and its corresponding roles in filopodium formation and tumor metastasis. Methods Immunoprecipitation and glutathione‐S‐transferase pull‐down assays were performed to examine the interaction between Fascin and acetyltransferase P300/CBP‐associated factor (PCAF), and immunofluorescence was used to investigate their colocalization. An in vitro acetylation assay was performed to identify Fascin acetylation sites by using mass spectrometry. A specific antibody against acetylated Fascin was generated and used to detect the PCAF‐mediated Fascin acetylation in esophageal squamous cell carcinoma (ESCC) cells using Western blotting by overexpressing and knocking down PCAF expression. An in vitro cell migration assay was performed, and a xenograft model was established to study in vivo tumor metastasis. Live‐cell imaging and fluorescence recovery after photobleaching were used to evaluate the function and dynamics of acetylated Fascin in filopodium formation. The clinical significance of acetylated Fascin and PCAF in ESCC was evaluated using immunohistochemistry. Results Fascin directly interacted and colocalized with PCAF in the cytoplasm and was acetylated at lysine 471 (K471) by PCAF. Using the specific anti‐AcK471‐Fascin antibody, Fascin was found to be acetylated in ESCC cells, and the acetylation level was consequently increased after PCAF overexpression and decreased after PCAF knockdown. Functionally, Fascin‐K471 acetylation markedly suppressed in vitro ESCC cell migration and in vivo tumor metastasis, whereas Fascin‐K471 deacetylation exhibited a potent oncogenic function. Moreover, Fascin‐K471 acetylation reduced filopodial length and density, and lifespan of ESCC cells, while its deacetylation produced the opposite effect. In the filipodium shaft, K471‐acetylated Fascin displayed rapid dynamic exchange, suggesting that it remained in its monomeric form owing to its weakened actin‐bundling activity. Clinically, high levels of AcK471‐Fascin in ESCC tissues were strongly associated with prolonged overall survival and disease‐free survival of ESCC patients. Conclusions Fascin interacts directly with PCAF and is acetylated at lysine 471 in ESCC cells. Fascin‐K471 acetylation suppressed ESCC cell migration and tumor metastasis by reducing filopodium formation through the impairment of its actin‐bundling activity.

Immune checkpoint inhibitor (ICI) therapy is generating remarkable responses in individuals with cancer, but only a small portion of individuals with breast cancer respond well. Here we report that tumor-derived Jagged1 is a key regulator of the tumor immune microenvironment. Jagged1 promotes tumorigenesis in multiple spontaneous mammary tumor models. Through Jagged1-induced Notch activation, tumor cells increase expression and secretion of multiple cytokines to help recruit macrophages into the tumor microenvironment. Educated macrophages crosstalk with tumor-infiltrating T cells to inhibit T cell proliferation and tumoricidal activity. In individuals with triple-negative breast cancer, a high expression level of Jagged1 correlates with increased macrophage infiltration and decreased T cell activity. Co-administration of an ICI PD-1 antibody with a Notch inhibitor significantly inhibits tumor growth in breast cancer models. Our findings establish a distinct signaling cascade by which Jagged1 promotes adaptive immune evasion of tumor cells and provide several possible therapeutic targets.

Cell-cycle-associated and expression-elevated protein in tumor (CREPT) functions as a cell cycle modulator that enhances the transcription of cyclin D1 by interacting with RNA polymerase II. CREPT has been identified to be overexpressed in various human cancer types; however, the expression and significance of CREPT in renal cell carcinoma (RCC) has remained largely elusive. In the present study, increased expression of CREPT was identified in 46.7% RCC tissues compared with adjacent normal tissue (31.1%; P=0.032) using immunohistochemistry. Furthermore, overexpression of CREPT was significantly associated with the Tumor-Node-Metastasis stage (χ2=11.967, P=0.001) and Fuhrman grade (χ2=15.453, P

Nuclear factor-kappa B (NF-κB) activation is critical for innate immune responses. However, cellular-intrinsic regulation of NF-κB activity during inflammatory diseases remains incompletely understood. Ubiquitin-like protein 4A (UBL4A, GdX) is a small adaptor protein involved in protein folding, biogenesis and transcription. Yet, whether GdX has a role during innate immune response is largely unknown. Methods: To investigate the involvement of GdX in innate immunity, we challenged GdX-deficient mice with lipopolysaccharides (LPS). To investigate the underlying mechanism, we performed RNA sequencing, real-time PCR, ELISA, luciferase reporter assay, immunoprecipitation and immunoblot analyses, flow cytometry, and structure analyses. To investigate whether GdX functions in inflammatory bowel disease, we generated dendritic cell (DC), macrophage (Mφ), epithelial-cell specific GdX-deficient mice and induced colitis with dextran sulfate sodium. Results: GdX enhances DC and Mφ-mediated innate immune defenses by positively regulating NF-κB signaling. GdX-deficient mice were resistant to LPS-induced endotoxin shock and DSS-induced colitis. DC- or Mφ- specific GdX-deficient mice displayed alleviated mucosal inflammation. The production of pro-inflammatory cytokines by GdX-deficient DCs and Mφ was reduced. Mechanistically, we found that tyrosine-protein phosphatase non-receptor type 2 (PTPN2, TC45) and protein phosphatase 2A (PP2A) form a complex with RelA (p65) to mediate its dephosphorylation whereas GdX interrupts the TC45/PP2A/p65 complex formation and restrict p65 dephosphorylation by trapping TC45. Conclusion: Our study provides a mechanism by which NF-κB signaling is positively regulated by an adaptor protein GdX in DC or Mφ to maintain the innate immune response. Targeting GdX could be a strategy to reduce over-activated immune response in inflammatory diseases.