SMAD3 promotes expression and activity of the androgen receptor in prostate cancer.

Overexpression of androgen receptor (AR) is the primary cause of castration-resistant prostate cancer, although mechanisms upregulating AR transcription in this context are not well understood. Our RNA-seq studies revealed that SMAD3 knockdown decreased levels of AR and AR target genes, whereas SMAD4 or SMAD2 knockdown had little or no effect. ChIP-seq analysis showed that SMAD3 knockdown decreased global binding of AR to chromatin. Mechanistically, we show that SMAD3 binds to intron 3 of the AR gene to promote AR expression. Targeting these binding sites by CRISPRi reduced transcript levels of AR and AR targets. In addition, ∼50% of AR and SMAD3 ChIP-seq peaks overlapped, and SMAD3 may also cooperate with or co-activate AR for AR target expression. Functionally, AR re-expression in SMAD3-knockdown cells partially rescued AR target expression and cell growth defects. The SMAD3 peak in AR intron 3 overlapped with H3K27ac ChIP-seq and ATAC-seq peaks in datasets of prostate cancer. AR and SMAD3 mRNAs were upregulated in datasets of metastatic prostate cancer and CRPC compared with primary prostate cancer. A SMAD3 PROTAC inhibitor reduced levels of AR, AR-V7 and AR targets in prostate cancer cells. This study suggests that SMAD3 could be targeted to inhibit AR in prostate cancer.

Histone demethylase JMJD1A in cancer progression and therapeutic resistance.

JMJD1A (also called lysine demethylase 3A [KDM3A]) belongs to the Jumonji C family of histone demethylases. It specifically removes the repressive mono- or di-methyl marks from histone H3 at lysine 9 and thus contributes to the activation of gene transcription. JMJD1A plays a key role in a variety of biological processes such as spermatogenesis, metabolism, sex determination, and stem cell activity. JMJD1A is upregulated in various types of cancers and can promote cancer development, progression, and therapeutic resistance. JMJD1A can epigenetically regulate the expression or activity of transcription factors such as c-Myc, androgen receptor (AR), estrogen receptor (ER), ß-catenin, and so on. Expression and activity of JMJD1A in cancer cells can be regulated at transcriptional, post-transcriptional, and post-translational levels. Targeting JMJD1A may repress the oncogenic transcription factors as a potential anticancer therapy.

Orally administered collagen peptide protects against UVB-induced skin aging through the absorption of dipeptide forms, Gly-Pro and Pro-Hyp.

Collagen hydrolysate is a well-known nutritional supplement for the improvement of healthy skin. Here, collagen peptide NS (CPNS) from fish scale was prepared, and its physicochemical properties were investigated. Gly-Pro was revealed as a representative low molecular weight peptide of CPNS, by performing prep-HPLC and LC-MS/MS. CPNS treatment attenuated matrix metalloproteinase-1 production and increased the synthesis of type 1 procollagen in HDF cells. After orally administering CPNS to rats, the plasma concentrations of Gly-Pro and Pro-Hyp increased dramatically. To examine the protective effects of CPNS against ultraviolet B (UVB)-induced photoaging in vivo, the dorsal skins of hairless mice were exposed to UVB and supplemented with CPNS for 12 weeks. The CPNS consumption significantly attenuated UVB-induced wrinkle formation, transepidermal water loss, and epidermis thickness, and increased skin hydration. Collectively, these results suggest that bioactive peptides of CPNS, Gly-Pro and Pro-Hyp, exert beneficial effects on skin health.

Conversion of glioma cells to glioma stem-like cells by angiocrine factors.

Glioma stem-like cells (GSCs) contribute to tumor initiation, progression, and therapeutic resistance, but their cellular origin remains largely unknown. Here, using a stem/progenitor cell-fate tracking reporter system in which eGFP is expressed by promoter of OCT4 that is activated in stem/progenitor cells, we demonstrate that eGFP-negative glioma cells (GCs) became eGFP-positive-GCs in both in vitro cultures and in vivo xenografts. These eGFP-positive-GCs exhibited GSC features and primarily localized to the perivascular region in tumor xenografts, similar to the existence of OCT4-expressing GCs in the perivascular region of human glioblastoma specimens. Angiocrine factors, including nitric oxide (NO), converted eGFP-negative-GCs into eGFP-positive-GCs. Mechanistically, NO signaling conferred GSC features to GCs by increasing OCT4 and NOTCH signaling via ID4. NO signaling blockade and a suicide gene induction prevented tumorigenicity with a decrease in eGFP-positive-GCs in the perivascular region. Taken together, our results reveal the molecular mechanism underlying GSCs generation by cancer cell dedifferentiation.

Verapamil augments carmustine- and irradiation-induced senescence in glioma cells by reducing intracellular reactive oxygen species and calcium ion levels.

Resistance to conventional therapies and frequent recurrence are the major obstacles to the treatment of high-grade gliomas, including glioblastoma. Thus, the development of new therapeutic strategies to overcome these obstacles is necessary to improve the treatment outcomes. In this study, we found that verapamil, a pan-adenosine triphosphate-binding cassette transporter and L-type voltage-dependent calcium channel inhibitor, sensitized U87MG glioma cells to carmustine- and irradiation-induced senescence. Furthermore, our results indicated that verapamil treatment, in combination with carmustine and irradiation, rendered U87MG glioma cells and several patient-derived glioma stem cells more sensitive to therapy-induced senescence than individual or dual-combination treatments. When investigating the underlying mechanism, we found that verapamil treatment markedly decreased intracellular reactive oxygen species and calcium ion levels. Reactive oxygen species reduction with N-acetylcysteine, a reactive oxygen species scavenger, rendered U87MG glioma cells more sensitive to carmustine and irradiation whereas the protein kinase C agonist, phorbol 12-myristate 13-acetate, mitigated the effects of carmustine and irradiation. Taken together, our results indicate that verapamil may be a potent therapeutic sensitizer for increasing the effectiveness of glioblastoma treatment.

Irradiation induces glioblastoma cell senescence and senescence-associated secretory phenotype.

Glioblastoma multiforme (GBM) is one of the most aggressive and fatal primary brain tumors in humans. The standard therapy for the treatment of GBM is surgical resection, followed by radiotherapy and/or chemotherapy. However, the frequency of tumor recurrence in GBM patients is very high, and the survival rate remains poor. Delineating the mechanisms of GBM recurrence is essential for therapeutic advances. Here, we demonstrate that irradiation rendered 17-20 % of GBM cells dead, but resulted in 60-80 % of GBM cells growth-arrested with increases in senescence markers, such as senescence-associated beta-galactosidase-positive cells, H3K9me3-positive cells, and p53-p21(CIP1)-positive cells. Moreover, irradiation induced expression of senescence-associated secretory phenotype (SASP) mRNAs and NFκB transcriptional activity in GBM cells. Strikingly, compared to injection of non-irradiated GBM cells into immune-deficient mice, the co-injection of irradiated and non-irradiated GBM cells resulted in faster growth of tumors with the histological features of human GBM. Taken together, our findings suggest that the increases in senescent cells and SASP in GBM cells after irradiation is likely one of main reasons for tumor recurrence in post-radiotherapy GBM patients.

Preparation of Two New Diasteromeric Chiral Stationary Phases Based on (+)-(18-Crown-6)-2,3,11,12-tetracarboxylic Acid and (R)- or (S)-1-(1-Naphthyl)ethylamine and Chiral Tethering Group Effect on the Chiral Recognition.

Two new diastereomeric chiral stationary phases (CSPs) based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid as a chiral tethering group and a Π-basic chiral unit such as (R)-1-(1-naphthyl)ethylamine (CSP 1) or (S)-1-(1-naphthyl)ethylamine (CSP 2) were prepared. The two CSPs were applied to the enantiomeric separation of N-(3,5-dinitrobenzoyl)-1-phenylalkylamines and N-(3,5-dinitrobenzoyl)-α-amino acid derivatives using 20% isopropyl alcohol in hexane as a normal mobile phase. To elucidate the effect of the two chiral units on the chiral recognition, the chiral recognition abilities of the two CSPs were compared with each other and with that of a CSP (CSP 3) based on (R)-1-(1-naphthyl)ethylamine. From the chromatographic chiral recognition results, (R)-1-(1-naphthyl)ethylamine and (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid constituting CSP 1 were concluded to show a cooperative ("matched") effect on the chiral recognition while (S)-1-(1-naphthyl)ethylamine and (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid constituting CSP 2 were concluded to show an uncooperative ("mismatched") effect on the chiral recognition. From these results, it was concluded that (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid can be successfully used as a chiral tethering group for the preparation of new CSPs.

Generation of reproductive transgenic pigs of a CRISPR-Cas9-based oncogene-inducible system by somatic cell nuclear transfer.

Alternative cancer models that are close to humans are required to create more valuable preclinical results during oncology studies. Here, a new onco-pig model via developing a CRISPR-Cas9-based Conditional Polycistronic gene expression Cassette (CRI-CPC) system to control the tumor inducing simian virus 40 large T antigen (SV40LT) and oncogenic HRASG12V . After conducting somatic cell nuclear transfer (SCNT), transgenic embryos were transplanted into surrogate mothers and five male piglets were born. Umbilical cord analysis confirmed that all piglets were transgenic. Two of them survived and they expressed a detectable green fluorescence. The test was made whether CRI-CPC models were naturally fertile and whether the CRI-CPC system was stably transferred to the offspring. By mating with a normal female pig, four offspring piglets were successfully produced. Among them, only three male piglets were transgenic. Finally, their applicability was tested as cancer models after transduction of Cas9 into fibroblasts from each CRI-CPC pig in vitro, resulting in cell acquisition of cancerous characteristics via the induction of oncogene expression. These results showed that our new CRISPR-Cas9-based onco-pig model was successfully developed.

Role of H3K9 demethylases in DNA double-strand break repair.

H3K9 demethylases can remove the repressive H3K9 methylation marks on histones to alter chromatin structure, gene transcription and epigenetic state of cells. By counteracting the function of H3K9 methyltransferases, H3K9 demethylases have been shown to play an important role in numerous biological processes, including diseases such as cancer. Recent evidence points to a key role for some H3K9 demethylases in the repair of DNA double-strand breaks (DSBs) via homologous recombination (HR) and/or non-homologous end joining (NHEJ) pathways. Mechanistically, H3K9 demethylases can upregulate the expression of DNA repair factors. They can also be recruited to the DNA damage sites and regulate the recruitment or function of DNA repair factors. Here, we will discuss the role and mechanisms of H3K9 demethylases in the regulation of DSB repair.

p300-Mediated Acetylation of Histone Demethylase JMJD1A Prevents Its Degradation by Ubiquitin Ligase STUB1 and Enhances Its Activity in Prostate Cancer.

The androgen receptor (AR) pathway plays a central role in the development of castration-resistant prostate cancer (CRPC). The histone demethylase JMJD1A has been shown to regulate activities of AR and c-Myc transcription factors and promote prostate cancer progression. Here, we report that JMJD1A protein stability is controlled by the ubiquitin ligase STUB1. High levels of JMJD1A were strongly correlated with low STUB1 levels in human CRPC specimens. STUB1 inhibited AR activity, AR-V7 levels, and prostate cancer cell growth partly through degradation of JMJD1A. Furthermore, the acetyltransferase p300 acetylated JMJD1A at lysine (K) 421, a modification that recruits the BET family member BRD4 to block JMJD1A degradation and promote JMJD1A recruitment to AR targets. Increased levels of both total and K421-acetylated JMJD1A were observed in prostate cancer cells as they developed resistance to the AR antagonist enzalutamide. Treatment of prostate cancer cells with either p300 or BET inhibitors destabilized JMJD1A, and enzalutamide-resistant prostate cancer cells were more sensitive than parental cells to these inhibitors. Together, our findings identify a critical role for acetylation of JMJD1A in regulating JMJD1A stability and AR activity in CRPC. These newly identified mechanisms controlling JMJD1A protein stability provide potential druggable targets to encourage the development of additional therapies for advanced prostate cancer. SIGNIFICANCE: Identification of mechanisms regulating JMJD1A protein stability reveals new strategies to destabilize JMJD1A and concomitantly inhibit AR activities as potential prostate cancer therapy.

Antifibrotic activity of triterpenoids from the aerial parts of Euscaphis japonica on hepatic stellate cells.

In the course of screening antifibrotic activity of natural products employing HSC-T6, a rat hepatic stellate cell line, as an in vitro assay system, the methanolic extract of aerial parts of Euscaphis japonica (Tunb.) Kantiz (Staphyleaceae) showed significant inhibitory activity on HSC proliferation. Activity-guided fractionation led to the isolation of four triterpenoids, friedeline (1), glut-5-en-ol (2), pomolic acid (3), and methylrotundate (4). Among the triterpenoids isolated, pomolic acid (3) significantly inhibited the proliferation of HSCs at concentrations 10 and 100 microM.

OCT4B Isoform Promotes Anchorage-Independent Growth of Glioblastoma Cells.

OCT4, also known as POU5F1 (POU domain class 5 transcription factor 1), is a transcription factor that acts as a master regulator of pluripotency in embryonic stem cells and is one of the reprogramming factors required for generating induced pluripotent stem cells. The human OCT4 encodes three isoforms, OCT4A, OCT4B, and OCT4B1, which are generated by alternative splicing. Currently, the functions and expression patterns of OCT4B remain largely unknown in malignancies, especially in human glioblastomas. Here, we demonstrated the function of OCT4B in human glioblastomas. Among the isoform of OCT4B, OCT4B-190 (OCT4B19kDa) was highly expressed in human glioblastoma stem cells and glioblastoma cells and was mainly detected in the cytoplasm rather than the nucleus. Overexpression of OCT4B19kDa promoted colony formation of glioblastoma cells when grown in soft agar culture conditions. Clinical data analysis revealed that patients with gliomas that expressed OCT4B at high levels had a poorer prognosis than patients with gliomas that expressed OCT4B at low levels. Thus, OCT4B19kDa may play a crucial role in regulating cancer cell survival and adaption in a rigid environment.

TP53 gain-of-function mutation promotes inflammation in glioblastoma.

Glioblastoma (GBM), the most severe and common brain tumor in adults, is characterized by multiple somatic mutations and aberrant activation of inflammatory responses. Immune cell infiltration and subsequent inflammation cause tumor growth and resistance to therapy. Somatic loss-of-function mutations in the gene encoding tumor suppressor protein p53 (TP53) are frequently observed in various cancers. However, numerous studies suggest that TP53 regulates malignant phenotypes by gain-of-function (GOF) mutations. Here we demonstrate that a TP53 GOF mutation promotes inflammation in GBM. Ectopic expression of a TP53 GOF mutant induced transcriptomic changes, which resulted in enrichment of gene signatures related to inflammation and chemotaxis. Bioinformatics analyses revealed that a gene signature, upregulated by the TP53 GOF mutation, is associated with progression and shorter overall survival in GBM. We also observed significant correlations between the TP53 GOF mutation signature and inflammation in the clinical database of GBM and other cancers. The TP53 GOF mutant showed upregulated C-C motif chemokine ligand 2 (CCL2) and tumor necrosis factor alpha (TNFA) expression via nuclear factor kappa B (NFκB) signaling, consequently increasing microglia and monocyte-derived immune cell infiltration. Additionally, TP53 GOF mutation and CCL2 and TNFA expression correlated positively with tumor-associated immunity in patients with GBM. Taken together, our findings suggest that the TP53 GOF mutation plays a crucial role in inflammatory responses, thereby deteriorating prognostic outcomes in patients with GBM.

Korean Red ginseng extract inhibits glioblastoma propagation by blocking the Wnt signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Korean Red ginseng extract (RG) is one of the most widely used traditional health functional food in Asia, which invigorates immunity and vital energy. RG have been suggested to inhibit proliferation, invasion, and inflammation in several cancer cell lines. Correspondingly, clinical studies have raised the possibility that RG could augment therapeutic efficacy in cancer patients. However, little is known about the anti-cancer effects of RG in glioblastoma (GBM), the most common and aggressive brain tumor for which effective therapeutic regimens need to be developed. AIM OF THIS STUDY: Here, we assessed the in vivo and in vitro anti-cancer properties of RG in a patient-derived xenograft mouse model and GBM stem cell (GSC) line. MATERIALS AND METHODS: We evaluated the anti-cancer effects of RG in patient-derived GBM xenograft mice with and without combined concurrent chemo- and radiation therapy (CCRT). Furthermore, we verified the in vitro effects of RG on the proliferation, cell death, and stem cell-like self-renewal capacity of cancer cells. Finally, we investigated the signaling pathway affected by RG, via which its anti-cancer effects were mediated. RESULTS: When combined with CCRT, RG impeded GBM progression by reducing cancer cell proliferation and ionized calcium-binding adapter molecule 1 (IBA1)-positive immune cell recruitment. The anti-cancer effects of RG were mediated by Rg3 and Rh2 ginsenosides. Rg3 promoted cell death while Rh2 did not. Furthermore, both Rg3 and Rh2 reduced cell viability and self-renewal capacity of GSCs by inhibiting Wnt/ß-catenin signaling. CONCLUSION: Therefore, our observations imply that RG could be applied to the GBM patients in parallel with CCRT to enhance therapeutic efficacy.

Ly6G+ inflammatory cells enable the conversion of cancer cells to cancer stem cells in an irradiated glioblastoma model.

Most glioblastomas frequently recur at sites of radiotherapy, but it is unclear if changes in the tumor microenvironment due to radiotherapy influence glioblastoma recurrence. Here, we demonstrate that radiation-induced senescent glioblastoma cells exhibit a senescence-associated secretory phenotype that functions through NFκB signaling to influence changes in the tumor microenvironment, such as recruitment of Ly6G+ inflammatory cells and vessel formation. In particular, Ly6G+ cells promote conversion of glioblastoma cells to glioblastoma stem cells (GSCs) through the NOS2-NO-ID4 regulatory axis. Specific inhibition of NFκB signaling in irradiated glioma cells using the IκBα super repressor prevents changes in the tumor microenvironment and dedifferentiation of glioblastoma cells. Treatment with Ly6G-neutralizing antibodies also reduces the number of GSCs and prolongs survival in tumor-bearing mice after radiotherapy. Clinically, a positive correlation exists between Ly6G+ cells and the NOS2-NO-ID4 regulatory axis in patients diagnosed with recurrent glioblastoma. Together, our results illustrate important roles for Ly6G+ inflammatory cells recruited by radiation-induced SASP in cancer cell dedifferentiation and tumor recurrence.

Inhibition of ID1-BMPR2 Intrinsic Signaling Sensitizes Glioma Stem Cells to Differentiation Therapy.

Purpose: Normal stem cells tightly control self-renewal and differentiation during development, but their neoplastic counterparts, cancer stem cells (CSCs), sustain tumorigenicity both through aberrant activation of stemness and evasion of differentiation. Although regulation of CSC stemness has been extensively studied, the molecular mechanisms suppressing differentiation remain unclear.Experimental Design: We performed in silico screening and in vitro validation studies through Western blotting, qRT-PCR for treatment of WNT and SHH signaling inhibitors, and BMP signaling inducer with control and ID1-overexpressing cells. We also performed in vivo drug treatment assays with Balb/c nude mice.Results: Inhibitor of differentiation 1 (ID1) abrogated differentiation signals from bone morphogenetic protein receptor (BMPR) signaling in glioblastoma stem cells (GSCs) to promote self-renewal. ID1 inhibited BMPR2 expression through miRNAs, miR-17 and miR-20a, which are transcriptional targets of MYC. ID1 increases MYC expression by activating WNT and SHH signaling. Combined pharmacologic blockade of WNT and SHH signaling with BMP treatment significantly suppressed GSC self-renewal and extended survival of tumor-bearing mice.Conclusions: Collectively, our results suggested that ID1 simultaneously regulates stemness through WNT and SHH signaling and differentiation through BMPR-mediated differentiation signaling in GSCs, informing a novel therapeutic strategy of combinatorial targeting of stemness and differentiation. Clin Cancer Res; 24(2); 383-94. ©2017 AACR.

Comparison of Cellular Transforming Activity of OCT4, NANOG, and SOX2 in Immortalized Astrocytes.

Embryonic stem cell factors-OCT4, NANOG, and SOX2-contribute to the maintenance of stem cell properties and malignant progression in various cancers, including glioblastoma. Although functional roles of each of these genes are well documented in stem cell and cancer biology, no study has directly compared their cellular transforming activity under same experimental conditions. In this study, we compared the cellular transforming activity of OCT4, NANOG, and SOX2 using human immortalized astrocytes cultured under serum-free stem cell culture conditions. We found that SOX2 exhibited the strongest transforming activities, such as cell proliferation, neurosphere formation, resistance to cytotoxic drug, and cell migration/invasion, which may be associated with the activation of the nuclear factor kappa B (NFκB) signaling pathway. Thus, OCT4, NANOG, and SOX2, known to be frequently activated in various cancers and cancer stem cells, may play a distinct role in the regulation of cellular transformation.

CD133 Regulates IL-1ß Signaling and Neutrophil Recruitment in Glioblastoma.

CD133, a pentaspan transmembrane glycoprotein, is generally used as a cancer stem cell marker in various human malignancies, but its biological function in cancer cells, especially in glioma cells, is largely unknown. Here, we demonstrated that forced expression of CD133 increases the expression of IL-1ß and its downstream chemokines, namely, CCL3, CXCL3 and CXCL5, in U87MG glioma cells. Although there were no apparent changes in cell growth and sphere formation in vitro and tumor growth in vivo, in vitro trans-well studies and in vivo tumor xenograft assays showed that neutrophil recruitment was markedly increased by the ectopic expression of CD133. In addition, the clinical relevance between CD133 expression and IL-1ß gene signature was established in patients with malignant gliomas. Thus, these results imply that glioma cells expressing CD133 are capable of modulating tumor microenvironment through the IL-1ß signaling pathway.

BRM270, a Compound from Natural Plant Extracts, Inhibits Glioblastoma Stem Cell Properties and Glioblastoma Recurrence.

Glioblastoma multiforme (GBM) is one of the most aggressive and lethal human brain tumors, and the median survival of patients with GBM is only 14 months. Glioblastoma stem cells (GSCs) are regarded as a main cause of GBM recurrence, because of their self-renewal and drug resistance properties. Therefore, targeting GSCs is an important therapeutic strategy for GBM. In this study, we show the effects of BRM270, a compound from natural plant extracts, on GSCs in vitro and GBM recurrence in vivo. BRM270 induced apoptotic cell death and inhibited cell growth and "stemness" both in vitro and in vivo. Combining BRM270 treatment with concurrent chemoradiotherapy (CCRT) dramatically increased mice survival and tumor growth inhibition. Taken together, our results suggested that BRM270 synergizes with CCRT as a therapeutic agent to target GSCs.

The ID1-CULLIN3 Axis Regulates Intracellular SHH and WNT Signaling in Glioblastoma Stem Cells.

Inhibitor of differentiation 1 (ID1) is highly expressed in glioblastoma stem cells (GSCs). However, the regulatory mechanism responsible for its role in GSCs is poorly understood. Here, we report that ID1 activates GSC proliferation, self-renewal, and tumorigenicity by suppressing CULLIN3 ubiquitin ligase. ID1 induces cell proliferation through increase of CYCLIN E, a target molecule of CULLIN3. ID1 overexpression or CULLIN3 knockdown confers GSC features and tumorigenicity to murine Ink4a/Arf-deficient astrocytes. Proteomics analysis revealed that CULLIN3 interacts with GLI2 and DVL2 and induces their degradation via ubiquitination. Consistent with ID1 knockdown or CULLIN3 overexpression in human GSCs, pharmacologically combined control of GLI2 and ß-CATENIN effectively diminishes GSC properties. A ID1-high/CULLIN3-low expression signature correlates with a poor patient prognosis, supporting the clinical relevance of this signaling axis. Taken together, a loss of CULLIN3 represents a common signaling node for controlling the activity of intracellular WNT and SHH signaling pathways mediated by ID1.