EZH2/hSULF1 axis mediates receptor tyrosine kinase signaling to shape cartilage tumor progression.

Chondrosarcomas are primary cancers of cartilaginous tissue and capable of alteration to highly aggressive, metastatic, and treatment-refractory states, leading to a poor prognosis with a five-year survival rate at 11 months for dedifferentiated subtype. At present, the surgical resection of chondrosarcoma is the only effective treatment, and no other treatment options including targeted therapies, conventional chemotherapies, or immunotherapies are available for these patients. Here, we identify a signal pathway way involving EZH2/SULF1/cMET axis that contributes to malignancy of chondrosarcoma and provides a potential therapeutic option for the disease. A non-biased chromatin immunoprecipitation sequence, cDNA microarray analysis, and validation of chondrosarcoma cell lines identified sulfatase 1 (SULF1) as the top EZH2-targeted gene to regulate chondrosarcoma progression. Overexpressed EZH2 resulted in downregulation of SULF1 in chondrosarcoma cell lines, which in turn activated cMET pathway. Pharmaceutical inhibition of cMET or genetically silenced cMET pathway significantly retards the chondrosarcoma growth and extends mice survival. The regulation of EZH2/SULF1/cMET axis were further validated in patient samples with chondrosarcoma. The results not only established a signal pathway promoting malignancy of chondrosarcoma but also provided a therapeutic potential for further development of effective target therapy to treat chondrosarcoma.

Growth-promoting function of the cGAS-STING pathway in triple-negative breast cancer cells.

The cGAS-STING axis is one of the key mechanisms guarding cells from pathogen invasion in the cytoplasmic compartment. Sensing of foreign DNA in the cytosol by the cGAS-STING axis triggers a stress cascade, culminating at stimulation of the protein kinase TBK1 and subsequently activation of inflammatory response. In cancer cells, aberrant metabolism of the genomic DNA induced by the hostile milieu of tumor microenvironment or stresses brought about by cancer therapeutics are the major causes of the presence of nuclear DNA in the cytosol, which subsequently triggers a stress response. However, how the advanced tumors perceive and tolerate the potentially detrimental effects of cytosolic DNA remains unclear. Here we show that growth limitation by serum starvation activated the cGAS-STING pathway in breast cancer cells, and inhibition of cGAS-STING resulted in cell death through an autophagy-dependent mechanism. These results suggest that, instead of being subject to growth inhibition, tumors exploit the cGAS-STING axis and turn it to a survival advantage in the stressful microenvironment, providing a new therapeutic opportunity against advanced cancer. Concomitant inhibition of the cGAS-STING axis and growth factor signaling mediated by the epidermal growth factor receptor (EGFR) synergistically suppressed the development of tumor organoids derived from primary tumor tissues of triple-negative breast cancer (TNBC). The current study unveils an unexpected function of the cGAS-STING axis in promoting cancer cell survival and the potential of developing the stress-responding pathway as a therapeutic target, meanwhile highlights the substantial concerns of enhancing the pathway's activity as a means of anti-cancer treatment.

Epigenetic Silencing of miR-9 Promotes Migration and Invasion by EZH2 in Glioblastoma Cells.

Glioblastoma (GBM) is the most common primary brain tumor in adults. Tumor invasion is the major reason for treatment failure and poor prognosis in GBM. Inhibiting migration and invasion has become an important therapeutic strategy for GBM treatment. Enhancer of zeste homolog 2 (EZH2) and C-X-C motif chemokine receptor 4 (CXCR4) have been determined to have important roles in the occurrence and development of tumors, but the specific relationship between EZH2 and CXCR4 expression in GBM is less well characterized. In this study, we report that EZH2 and CXCR4 were overexpressed in glioma patients. Furthermore, elevated EZH2 and CXCR4 were correlated with shorter disease-free survival. In three human GBM cell lines, EZH2 modulated the expression of miR-9, which directly targeted the oncogenic signaling of CXCR4 in GBM. The ectopic expression of miR-9 dramatically inhibited the migratory capacity of GBM cells in vitro. Taken together, our results indicate that miR-9, functioning as a tumor-suppressive miRNA in GBM, is suppressed through epigenetic silencing by EZH2. Thus, miR-9 may be an attractive target for therapeutic intervention in GBM.

Eicosapentaenoic acids enhance chemosensitivity through connexin 43 upregulation in murine melanoma models.

Chemotherapy is now in common use for the treatment of tumors; however, with tumor growth retardation comes the severe side effects that occur after a chemotherapy cycle. Eicosapentaenoic acids (EPA) used in combination with chemotherapy has an additive effects and provides a rationale for using EPA in tandem with chemotherapy. To improve the efficacy and safety of this combination therapy, a further understanding that EPA modulates with the tumor microenvironment is necessary. Connexin 43 (Cx43) is involved in enhancing chemosensitivity that was suppressed in a tumor microenvironment. We aim to investigate the role of EPA in chemosensitivity in murine melanoma by inducing Cx43 expression. The dose-dependent upregulation of Cx43 expression and gap junction intercellular communication were observed in B16F10 cells after EPA treatment. Furthermore, EPA significantly increased the expression levels of mitogen-activated protein kinases (MAPK) signaling pathways. The EPA-induced Cx43 expression was reduced after MAPK inhibitors. Knockdown Cx43 in B16F10 cells reduced the therapeutic effects of combination therapy (EPA plus 5-Fluorouracil). Our results demonstrate that the treatment of EPA is a tumor induced Cx43 gap junction communication and enhances the combination of EPA and chemotherapeutic effects.

Fibroblast growth factors: Potential novel targets for regenerative therapy of osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disorder and is the leading cause of disability of people, which negatively impact people's physical and mental health. Although OA causes great socioeconomic burden and individual suffering, no effective treatment options are provided so far. This is partially resulted from poor regenerative activity of articular cartilage and our incomplete understanding of the underlying mechanism of OA. Traditional drug therapies such as acetaminophen and opioids are effective in relieving pain but do not reverse cartilage damage and are often associated with adverse events. Therefore, it is necessary to find effective OA drugs. In recent years, novel regenerative therapies have received much attention because they can effectively promote tissue repair and regeneration. The fibroblast growth factor (FGF) signaling has been suggested to involve in cartilage homeostasis for decades. The current research shows that sprifermin/recombinant FGF18 significantly reduces the loss of cartilage thickness and volume without serious side effects, thus warrants a continued research for potential new medications of OA. This review mainly highlights the current research progress on FGFs and FGF receptors as a potential therapeutic target for OA.

FOXA1 transcriptionally up-regulates cyclin B1 expression to enhance chondrosarcoma progression.

Chondrosarcoma is a malignant and common bone tumor that is highly resistant to radiation and chemotherapy. At this moment, amputation surgery is the only option which unfortunately has serious impact to daily lives of the patients. Thus, there is an urgent need to understand causative molecular mechanisms underlying the disease for more accurate prognosis and more effective targeted treatment. In the current study, we identify the transcription factor FOXA1 through cDNA microarray screening comparing normal versus chondrosarcoma cells and investigate the mechanisms underlying its function in chondrosarcoma development. We show that FOXA1 enhances expression of the cyclin B1 gene, which in turn drives cell cycle progression through G2-M transition thus promotes cell cycle progression of chondrosarcoma cells.

Rhodobacter sphaeroides Extract Lycogen™ Attenuates Testosterone-Induced Benign Prostate Hyperplasia in Rats.

Benign prostate hyperplasia (BPH) is one of the most common urological problems in mid-aged to elderly men. Risk factors of BPH include family history, obesity, type 2 diabetes, and high oxidative stress. The main medication classes for BPH management are alpha blockers and 5α-reductase inhibitors. However, these conventional medicines cause adverse effects. Lycogen™, extracted from Rhodobacter sphaeroides WL-APD911, is an anti-oxidant and anti-inflammatory compound. In this study, the effect of Lycogen™ was evaluated in rats with testosterone-induced benign prostate hyperplasia (BPH). Testosterone injections and Lycogen™ administration were carried out for 28 days, and body weights were recorded twice per week. The testosterone injection successfully induced a prostate enlargement. BPH-induced rats treated with different doses of Lycogen™ exhibited a significantly decreased prostate index (PI). Moreover, the Lycogen™ administration recovered the histological abnormalities observed in the prostate of BPH rats. In conclusion, these findings support a dose-dependent preventing effect of Lycogen™ on testosterone-induced BPH in rats and suggest that Lycogen™ may be favorable to the prevention and management of benign prostate hyperplasia.

YY1 and HDAC9c transcriptionally regulate p38-mediated mesenchymal stem cell differentiation into osteoblasts.

Mesenchymal stem cells (MSCs) have a high self-renewal potential and can differentiate into various types of cells, including adipocytes, osteoblasts, and chondrocytes. Previously, we reported that the enhancer of zeste homolog 2 (EZH2), the catalytic component of the Polycomb-repressive complex 2, and HDAC9c mediate the osteogenesis and adipogenesis of MSCs. In the current study, we identify the role of p38 in osteogenic differentiation from a MAPK antibody array screen and investigate the mechanisms underlying its transcriptional regulation. Our data show that YY1, a ubiquitously expressed transcription factor, and HDAC9c coordinate p38 transcriptional activity to promote its expression to facilitate the osteogenic potential of MSCs. Our results show that p38 mediates osteogenic differentiation, and this has significant implications in bone-related diseases, bone tissue engineering, and regenerative medicine.

Immunohistochemical biomarkers and volumetric parameters for predicting radiotherapy-based outcomes in patients with p16-negative pharyngeal cancer.

BACKGROUND: This study determined the prognostic effects of immunohistochemical biomarkers and volumetric parameters predicting radiotherapy-based treatment in patients with p16-negative squamous cell carcinoma of the oropharynx or hypopharynx. RESULTS: VEGF immunoreactivity > 2 and GLUT1 overexpression were prognostic factors for lower cause-specific survival. Moreover, both factors were associated with lower disease-free survival. The predictors of lower primary relapse-free survival were VEGF immunoreactivity > 2 and CT-based gross tumor volume > 16 mL. MATERIALS AND METHODS: Immunohistochemical biomarkers in pretreatment biopsy specimens from 60 patients with p16-negative cancer were analyzed using tissue microarrays. Computed tomography (CT)-based and biological tumor volumes were retrieved through fluorodeoxyglucose positron emission tomography-CT. Correlations of cause-specific, disease-free, and primary relapse-free survival with volumetric parameters and the immunohistochemical biomarker score were investigated. CONCLUSIONS: For patients with p16-negative pharyngeal cancer receiving radiotherapy, treatment outcomes can be stratified by VEGF and GLUT1 expression and CT-based gross tumor volume.

Immunohistochemical overexpression of hypoxia-induced factor 1α associated with slow reduction in 18fluoro-2-deoxy-D-glucose uptake for chemoradiotherapy in patients with pharyngeal cancer.

BACKGROUND: This study examined genomic factors associated with a reduction in 18fluoro-2-deoxy-D-glucose (FDG) uptake during positron emission tomography-computed tomography (PET-CT) for definitive chemoradiotherapy (CRT) in patients with pharyngeal cancer. METHODS: The pretreatment and interim PET-CT images of 25 patients with advanced pharyngeal cancers receiving definitive CRT were prospectively evaluated. The maximum standardized uptake value (SUVmax) of the interim PET-CT and the reduction ratio of the SUVmax (SRR) between the two images were measured. Genomic data from pretreatment incisional biopsy specimens (SLC2A1, CAIX, VEGF, HIF1A, BCL2, Claudin-4, YAP1, MET, MKI67, and EGFR) were analyzed using tissue microarrays. Differences in FDG uptake and SRRs between tumors with low and high gene expression were examined using the Mann-Whitney test. Cox regression analysis was performed to examine the effects of variables on local control. RESULTS: The SRR of the primary tumors (SRR-P) was 0.59 ± 0.31, whereas the SRR of metastatic lymph nodes (SRR-N) was 0.54 ± 0.32. Overexpression of HIF1A was associated with a high iSUVmax of the primary tumor (P < 0.001) and neck lymph node (P = 0.04) and a low SRR-P (P = 0.02). Multivariate analysis revealed that patients who had tumors with low SRR-P or high HIF1A expression levels showed inferior local control. CONCLUSION: In patients with pharyngeal cancer requiring CRT, HIF1A overexpression was positively associated with high interim SUVmax or a slow reduction in FDG uptake. Prospective trials are needed to determine whether the local control rate can be stratified using the HIF1A level as a biomarker and SRR-P.

Enhancer of Zeste Homolog 2 and Histone Deacetylase 9c Regulate Age-Dependent Mesenchymal Stem Cell Differentiation into Osteoblasts and Adipocytes.

Mesenchymal stem cells (MSCs) are multipotent precursors that can undergo multilineage differentiation, including osteogenesis and adipogenesis, which are two mutually exclusive events. Previously, we demonstrated that enhancer of zeste homolog 2 (EZH2), the catalytic component of the Polycomb-repressive complex 2, mediates epigenetic silencing of histone deacetylase 9c (HDAC9c) in adipocytes but not in osteoblasts and that HDAC9c accelerates osteogenesis while attenuating adipogenesis of MSCs through inactivation of peroxisome proliferator-activated receptor gamma 2 activity. Importantly, disrupting the balance between adipogenesis and osteogenesis can lead to age-associated bone loss (osteoporosis) and obesity. Here, we investigated the relationship between age, and osteogenic and adipogenic differentiation potential of MSCs by comparing EZH2 and HDAC9c expression in osteoblasts and adipocytes of both human and mice origins to determine whether the EZH2-HDAC9c axis regulates age-associated osteoporosis and obesity. Our findings indicated that a decline in HDAC9c expression over time was accompanied by increased EZH2 expression and suggested that a therapeutic intervention for age-associated osteoporosis and obesity may be feasible by targeting the EZH2-HDAC9c axis. Stem Cells 2016;34:2183-2193.

The Functional Haplotypes of CHRM3 Modulate mRNA Expression and Associate with Bladder Cancer among a Chinese Han Population in Kaohsiung City.

Bladder cancer is one of the major cancer types and both environmental factors and genetic background play important roles in its pathology. Kaohsiung is a high industrialized city in Taiwan, and here we focused on this region to evaluate the genetic effects on bladder cancer. Muscarinic acetylcholine receptor M3 (CHRM3) was reported as a key receptor in different cancer types. CHRM3 is located at 1q42-43 which was reported to associate with bladder cancer. Our study attempted to delineate whether genetic variants of CHRM3 contribute to bladder cancer in Chinese Han population in south Taiwan. Five selected SNPs (rs2165870, rs10802789, rs685550, rs7520974, and rs3738435) were genotyped for 30 bladder cancer patients and 60 control individuals and genetic association studies were performed. Five haplotypes (GTTAT, ATTGT, GCTAC, ACTAC, and ACCAC) were found significantly associated with low CHRM3 mRNA level and contributed to increased susceptibility of bladder cancer in Kaohsiung city after rigid 10000 consecutive permutation tests. To our knowledge, this is the first genetic association study that reveals the genetic contribution of CHRM3 gene in bladder cancer etiology.

Sequence variants of ADIPOQ and association with type 2 diabetes mellitus in Taiwan Chinese Han population.

Diabetes is a serious global health problem. Large-scale genome-wide association studies identified loci for type 2 diabetes mellitus (T2DM), including adiponectin (ADIPOQ) gene and transcription factor 7-like 2 (TCF7L2), but few studies clarified the effect of genetic polymorphisms of ADIPOQ and TCF7L2 on risk of T2DM. We attempted to elucidate association between T2DM and polymorphic variations of both in Taiwan's Chinese Han population, with our retrospective case-control study genotyping single nucleotide polymorphisms (SNPs) in ADIPOQ and TCF7L2 genes both in 149 T2DM patients and in 139 healthy controls from Taiwan. Statistical analysis gauged association of these polymorphisms with risk of T2DM to show ADIPOQ rs1501299 polymorphism variations strongly correlated with T2DM risk (P = 0.042), with rs2241766 polymorphism being not associated with T2DM (P = 0.967). However, both polymorphisms rs7903146 and rs12255372 of TCF7L2 were rarely detected in Taiwanese people. This study avers that ADIPOQ rs1501299 polymorphism contributes to risk of T2DM in the Taiwanese population.

EZH2: a pivotal regulator in controlling cell differentiation.

Epigenetic regulation plays an important role in stem cell self-renewal, maintenance and lineage differentiation. The epigenetic profiles of stem cells are related to their transcriptional signature. Enhancer of Zeste homlog 2 (EZH2), a catalytic subunit of epigenetic regulator Polycomb repressive complex 2 (PRC2), has been shown to be a key regulator in controlling cellular differentiation. EZH2 is a histone methyltransferase that not only methylates histone H3 on Lys 27 (H3K27me3) but also interacts with and recruits DNA methyltransferases to methylate CpG at certain EZH2 target genes to establish firm repressive chromatin structures, contributing to tumor progression and the regulation of development and lineage commitment both in embryonic stem cells (ESCs) and adult stem cells. In addition to its well-recognized epigenetic gene silencing function, EZH2 also directly methylates nonhistone targets such as the cardiac transcription factor, GATA4, resulting in attenuated GATA4 transcriptional activity and gene repression. This review addresses recent progress toward the understanding of the biological functions and regulatory mechanisms of EZH2 and its targets as well as their roles in stem cell maintenance and cell differentiation.

Nuclear ErbB2 enhances translation and cell growth by activating transcription of ribosomal RNA genes.

Aberrant regulation of rRNA synthesis and translation control can facilitate tumorigenesis. The ErbB2 growth factor receptor is overexpressed in many human tumors and has been detected in the nucleus, but the role of nuclear ErbB2 is obscure. In this study, we defined a novel function of nuclear ErbB2 in enhancing rRNA gene transcription by RNA polymerase-I (RNA Pol I). Nuclear ErbB2 physically associates with ß-actin and RNA Pol I, coinciding with active RNA Pol I transcription sites in nucleoli. RNA interference-mediated knockdown of ErbB2 reduced pre-rRNA and protein synthesis. In contrast, wild-type ErbB2 augmented pre-rRNA level, protein production, and cell size/cell growth, but not by an ErbB2 mutant that is defective in nuclear translocation. Chromatin immunoprecipitation assays revealed that ErbB2 enhances binding of RNA Pol I to rDNA. In addition, ErbB2 associated with rDNA, RNA Pol I, and ß-actin, suggesting how it could stimulate rRNA production, protein synthesis, and increased cell size and cell growth. Finally, ErbB2-potentiated RNA Pol I transcription could be stimulated by ligand and was not substantially repressed by inhibition of PI3-K and MEK/ERK (extracellular signal regulated kinase), the main ErbB2 effector signaling pathways. Together, our findings indicate that nuclear ErbB2 functions as a regulator of rRNA synthesis and cellular translation, which may contribute to tumor development and progression.

Myocyte enhancer factor-2 interacting transcriptional repressor (MITR) is a switch that promotes osteogenesis and inhibits adipogenesis of mesenchymal stem cells by inactivating peroxisome proliferator-activated receptor gamma-2.

EZH2, a catalytic subunit of Polycomb-repressive complex 2 (PRC2), is a histone lysine methyltransferase that methylates lysine 27 of histone H3, resulting in gene silencing. It has been shown that EZH2 plays a pivotal role in fostering self-renewal and inhibiting the differentiation of embryonic stem cells. Mesenchymal stem cells (MSCs) can be induced to differentiate into adipogenic and osteogenic lineages, which are mutually exclusive. However, it is not clear whether the molecular events of EZH2-mediated epigenetic silencing may coordinate differentiation between osteoblasts and adipocytes. Disruption of the balance between adipogenesis and osteogenesis is associated with many diseases; thus, identifying a switch that determines the fate of MSC is critical. In this study, we used EZH2-ChIP-on-chip assay to identify differential EZH2 targets in the two differentiation stages on a genome-wide scale. After validating the targets, we found that myocyte enhancer factor-2 interacting transcriptional repressor (MITR)/HDAC9c was expressed in osteoblasts and greatly decreased in adipocytes. We demonstrated that MITR plays a crucial role in the acceleration of MSC osteogenesis and attenuation of MSC adipogenesis through interaction with peroxisome proliferator-activated receptor (PPAR) γ-2 in the nucleus of osteoblasts, which interrupts PPARγ-2 activity and prevents adipogenesis. Together, our results demonstrated that MITR plays a master switch role to balance osteogenic and adipogenic differentiation of MSCs through regulation of PPARγ-2 transcriptional activity.

CDK1-dependent phosphorylation of EZH2 suppresses methylation of H3K27 and promotes osteogenic differentiation of human mesenchymal stem cells.

Enhancer of zeste homologue 2 (EZH2) is the catalytic subunit of Polycomb repressive complex 2 (PRC2) and catalyses the trimethylation of histone H3 on Lys 27 (H3K27), which represses gene transcription. EZH2 enhances cancer-cell invasiveness and regulates stem cell differentiation. Here, we demonstrate that EZH2 can be phosphorylated at Thr 487 through activation of cyclin-dependent kinase 1 (CDK1). The phosphorylation of EZH2 at Thr 487 disrupted EZH2 binding with the other PRC2 components SUZ12 and EED, and thereby inhibited EZH2 methyltransferase activity, resulting in inhibition of cancer-cell invasion. In human mesenchymal stem cells, activation of CDK1 promoted mesenchymal stem cell differentiation into osteoblasts through phosphorylation of EZH2 at Thr 487. These findings define a signalling link between CDK1 and EZH2 that may have an important role in diverse biological processes, including cancer-cell invasion and osteogenic differentiation of mesenchymal stem cells.

Regulation of tumor angiogenesis by EZH2.

Although VEGF-targeted therapies are showing promise, new angiogenesis targets are needed to make additional gains. Here, we show that increased Zeste homolog 2 (EZH2) expression in either tumor cells or in tumor vasculature is predictive of poor clinical outcome. The increase in endothelial EZH2 is a direct result of VEGF stimulation by a paracrine circuit that promotes angiogenesis by methylating and silencing vasohibin1 (vash1). Ezh2 silencing in the tumor-associated endothelial cells inhibited angiogenesis mediated by reactivation of VASH1, and reduced ovarian cancer growth, which is further enhanced in combination with ezh2 silencing in tumor cells. Collectively, these data support the potential for targeting ezh2 as an important therapeutic approach.

Downregulation of microRNA miR-520h by E1A contributes to anticancer activity.

The leading cause of death in cancer patients is cancer metastasis, for which there is no effective treatment. MicroRNAs (miRNA) have been shown to play a significant role in cancer metastasis through regulation of gene expression. The adenovirus type 5 E1A (E1A) is associated with multiple tumor-suppressing activities including the inhibition of metastasis, and E1A gene therapies have been tested in several clinical trials. However, the mechanisms involved in E1A-mediated tumor-suppressing activities are not yet completely defined. Here, we showed that E1A downregulated the expression of the miRNA miR-520h, which was critical for E1A-mediated cancer cell mobility and in vitro invasion activity. In addition, we identified a signal cascade, namely, E1A-->miRNA-520h-->PP2A/C-->IkappaB kinase-->NF-kappaB-->Twist, in which E1A inhibited the expression of Twist through downregulation of miR-520h and the signal cascade. Our results indicated a functional link between miR-520h and tumorigenicity/invasive ability and provided a new insight into the role of E1A-mediated miRNA regulation in tumor suppression. Therefore, the results identified a new cascade of E1A-mediated tumor suppression activity via downregulation of miRNA-520h expression.