Structures/cytotoxicity/selectivity relationship of natural steroidal saponins against GSCs and primary mechanism of tribulosaponin A.

Glioblastoma multiform (GBM) is the highly aggressive brain tumor with poor prognosis. Glioma stem cells (GSCs), small population of cancer cells that exist in GBM tissues, resistant to chemotherapy and radiotherapy and usually driving GBM recurrence, have been developed as effective therapeutic target. Steroidal saponins are one of important resources for anti-tumor agent and may be benefited to selectively clear GSCs. In this report, total of 97 natural steroidal saponins were investigated the relationship among structures/cytotoxicity/selectivity against GSCs, glioma cell lines and human untransformed cells, and revealed that tribulosaponin A was the most potent compound. Further investigation suggested that tribulosaponin A up-regulated the expression of NCF1 and NOX1 to accumulate ROS for triggering apoptosis in GSCs, but not in untransformed cells, and it was further supported by the assay that N-acetyl-l-cysteine (NAC) clearing ROS delayed GSCs apoptosis. Besides, tribulosaponin A damaged GSCs recapturing tumor spheres formation.

Glioma stem cell (GSC)-derived autoschizis-like products confer GSC niche properties involving M1-like tumor-associated macrophages.

Spontaneous necrosis is a defining feature of glioblastomas (GBMs), the most malignant glioma. Despite its strong correlations with poor prognosis, it remains unclear whether necrosis could be a possible cause or mere consequence of glioma progression. Here we isolated a particular fraction of necrotic products spontaneously arising from glioma cells, morphologically and biochemically defined as autoschizis-like products (ALPs). When administered to granulocyte macrophage colony-stimulating factor (GM-CSF)-primed bone marrow-derived macrophage/dendritic cells (Mφ/DCs), ALPs were found to be specifically engulfed by Mφs expressing a tumor-associated macrophage (TAM) marker CD204. ALPs from glioma stem cells (GSCs) had higher activity for the TAM development than those from non-GSCs. Of note, expression of the Il12b gene encoding a common subunit of IL-12/23 was upregulated in ALPs-educated Mφs. Furthermore, IL-12 protein evidently enhanced the sphere-forming activity of GBM patient-derived cells, although interestingly IL-12 is generally recognized as an antitumoral M1-Mφ marker. Finally, in silico analysis of The Cancer Genome Atlas (TCGA) transcriptome data of primary and recurrent GBMs revealed that higher expression of these IL-12 family genes was well correlated with more infiltration of M1-type TAMs and closely associated with poorer prognosis in recurrent GBMs. Our results highlight a role of necrosis in GSC-driven self-beneficial niche construction and glioma progression, providing important clues for developing new therapeutic strategies against gliomas.

The E3 ubiquitin ligase WWP2 facilitates RUNX2 protein transactivation in a mono-ubiquitination manner during osteogenic differentiation.

Poly-ubiquitination-mediated RUNX2 degradation is an important cause of age- and inflammation-related bone loss. NEDD4 family E3 ubiquitin protein ligases are thought to be the major regulators of RUNX2 poly-ubiquitination. However, we observed a mono-ubiquitination of RUNX2 that was catalyzed by WWP2, a member of the NEDD4 family of E3 ubiquitin ligases. WWP2 has been reported to catalyze the mono-ubiquitination of Goosecoid in chondrocytes, facilitating craniofacial skeleton development. In this study, we found that osteogenic differentiation of mesenchymal stem cells promoted WWP2 expression and nuclear accumulation. Knockdown of Wwp2 in mesenchymal stem cells and osteoblasts led to significant deficiencies of osteogenesis, including decreased mineral deposition and down-regulation of osteogenic marker genes. Co-immunoprecipitation experiments showed the interaction of WWP2 with RUNX2 in vitro and in vivo Mono-ubiquitination by WWP2 leads to RUNX2 transactivation, as evidenced by the wild type of WWP2, but not its ubiquitin ligase-dead mutant, augmenting RUNX2-reponsive reporter activity. Moreover, deletion of WWP2-dependent mono-ubiquitination resulted in striking defects of RUNX2 osteoblastic activity. In addition, ectopic expression of the constitutively active type 1A bone morphogenetic protein receptor enhanced WWP2-dependent RUNX2 ubiquitination and transactivation, demonstrating a regulatory role of bone morphogenetic protein signaling in the WWP2-RUNX2 axis. Taken together, our results provide evidence that WWP2 serves as a positive regulator of osteogenesis by augmenting RUNX2 transactivation in a non-proteolytic mono-ubiquitination manner.

c-Rel Regulates Inscuteable Gene Expression during Mouse Embryonic Stem Cell Differentiation.

Inscuteable (Insc) regulates cell fate decisions in several types of stem cells. Although it is recognized that the expression levels of mouse INSC govern the balance between symmetric and asymmetric stem cell division, regulation of mouse Insc gene expression remains poorly understood. Here, we showed that mouse Insc expression transiently increases at an early stage of differentiation, when mouse embryonic stem (mES) cells differentiate into bipotent mesendoderm capable of producing both endoderm and mesoderm in defined culture conditions. We identified the minimum transcriptional regulatory element (354 bases) that drives mouse Insc transcription in mES cells within a region >5 kb upstream of the mouse Insc transcription start site. We found that the transcription factor reticuloendotheliosis oncogene (c-Rel) bound to the minimum element and promoted mouse Insc expression in mES cells. In addition, short interfering RNA-mediated knockdown of either mouse INSC or c-Rel protein decreased mesodermal cell populations without affecting differentiation into the mesendoderm or endoderm. Furthermore, overexpression of mouse INSC rescued the mesoderm-reduced phenotype induced by knockdown of c-Rel. We propose that regulation of mouse Insc expression by c-Rel modulates cell fate decisions during mES cell differentiation.

Overexpression of goosecoid homeobox is associated with chemoresistance and poor prognosis in ovarian carcinoma.

Ovarian carcinoma is the most lethal cancer among all gynecological malignancies due to recurrence through chemoresistance. The aim of the present study was to identify new biomarkers to predict chemoresistance and prognosis in ovarian carcinomas. The mRNA expression by qRT-PCR was examined in 60 ovarian serous carcinomas for selected genes from the screening by PCR array focusing on apoptosis, epithelial-to-mesenchymal transition and cancer pathways. The clinical impact was assessed by analyzing the correlation between gene expression and clinicopathological variables. Further validation with immunohistochemistry was performed with 75 cases of serous carcinomas. The chemoresistance was significantly associated with high expression of FOS, GSC, SNAI1, TERT and TNFRSF10D, and low expression of CDKN1A, TNFRSF10A, TNFRSF10C and TRAF1 (p<0.05, t-test). Low expression of TRAF1 and high expression of E2F1, FOS, TERT and GSC were significantly associated with advanced clinical stage (p<0.05, χ2-test). Lymph node metastasis was significantly associated with high expression of GSC. The upregulation group of TERT, GSC, NOTCH1 and SNAI1, and downregulation group of TRAF1 were significantly associated with poor overall survival (p<0.05, log-rank test). On further validation with immunohistochemistry, overexpression of goosecoid homeobox (GSC) was associated with poor overall survival. The results suggest that GSC is the most potential biomarker of drug response and poor prognosis in ovarian serous carcinomas.

Chronic ethanol exposure increases goosecoid (GSC) expression in human embryonic carcinoma cell differentiation.

Fetal alcohol spectrum disorder (FASD) is a set of developmental malformations caused by excess alcohol consumption during pregnancy. Using an in vitro system, we examined the role that chronic ethanol (EtOH) exposure plays in gene expression changes during the early stage of embryonic differentiation. We demonstrated that EtOH affected the cell morphology, cell cycle progression and also delayed the down-regulation of OCT4 and NANOG during differentiation. Gene expression profiling and pathway analysis demonstrated that EtOH deregulates many genes and pathways that are involved in early embryogenesis. Follow-up analyzes revealed that EtOH exposure to embryoid bodies (EBs) induced the expression of an organizer-specific gene, goosecoid (GSC), in comparison to controls. Moreover, EtOH treatment altered several important genes that are involved in embryonic structure formation, nervous system development, and placental and embryonic vascularization, which are all common processes that FASD can disrupt. Specifically, EtOH treatment let to a reduction in ALDOC, ENO2 and CDH1 expression, whereas EtOH treatment induced the expression of PTCH1, EGLN1, VEGFA and DEC2 in treated EBs. We also found that folic acid (FA) treatment was able to correct the expression of the majority of genes deregulated by EtOH exposure during early embryo development. Finally, the present study identified a gene set including GSC, which was deregulated by EtOH exposure that may contribute to the etiology of fetal alcohol syndrome (FAS). We also reported that EtOH-induced GSC expression is mediated by Nodal signaling, which may provide a new avenue for analyzing the molecular mechanisms behind EtOH teratogenicity in FASD individuals.

The function of heterodimeric AP-1 comprised of c-Jun and c-Fos in activin mediated Spemann organizer gene expression.

BACKGROUND: Activator protein-1 (AP-1) is a mediator of BMP or FGF signaling during Xenopus embryogenesis. However, specific role of AP-1 in activin signaling has not been elucidated during vertebrate development. METHODOLOGY/PRINCIPAL FINDINGS: We provide new evidence showing that overexpression of heterodimeric AP-1 comprised of c-jun and c-fos (AP-1(c-Jun/c-Fos)) induces the expression of BMP-antagonizing organizer genes (noggin, chordin and goosecoid) that were normally expressed by high dose of activin. AP-1(c-Jun/c-Fos) enhanced the promoter activities of organizer genes but reduced that of PV.1, a BMP4-response gene. A loss of function study clearly demonstrated that AP-1(c-Jun/c-Fos) is required for the activin-induced organizer and neural gene expression. Moreover, physical interaction of AP-1(c-Jun/c-Fos) and Smad3 cooperatively enhanced the transcriptional activity of goosecoid via direct binding on this promoter. Interestingly, Smad3 mutants at c-Jun binding site failed in regulation of organizer genes, indicating that these physical interactions are specifically necessary for the expression of organizer genes. CONCLUSIONS/SIGNIFICANCE: AP-1(c-Jun/c-Fos) plays a specific role in organizer gene expression in downstream of activin signal during early Xenopus embryogenesis.

Regulatory expression of Brachyury and Goosecoid in P19 embryonal carcinoma cells.

Mouse P19 embryonal carcinoma cells can differentiate into various cell types depending on culture conditions. Here we show that the expression of the mesodermal genes Brachyury (Bra) and Goosecoid (Gsc) are under regulatory control in P19 cells. When P19 cells were cultured in a tissue culture dish in the presence of serum, Bra and Gsc were unexpectedly expressed. Expression of Bra and Gsc was greatly reduced with culture time, and expression levels at 144 h of culture were below 25% those at 48 h of culture. Members of the Tgf-beta family such as Activin and Nodal have been known to up-regulate expression of mesodermal genes. Treatment with SB431542, an Alk4/5/7 inhibitor, decreased Bra and Gsc in a dose-dependent manner, whereas it induced the expression of the neuroectodermal genes Mash-1 and Pax-6. Quantitative RT-PCR and dsRNAi transfection indicated Nodal as a possible ligand responsible for the regulation of Bra and Gsc. In addition, exogenous Nodal increased expression of Bra and Gsc in a dose-dependent manner. Serum concentration in culture medium positively related to expression of Nodal, Bra, Gsc, and Cripto, which encodes a membrane-tethered protein required for Nodal signaling. Addition of the culture supernatant of P19 cells at 144 h of culture to medium decreased expression of these genes. The present study reveals that stimulation and inhibition of the Nodal pathway increases mesodermal genes and neuroectodermal genes, respectively, indicating the importance of control of Nodal and Cripto expression for mesodermal formation and neurogenesis.

Sumoylation differentially regulates Goosecoid-mediated transcriptional repression.

Goosecoid (Gsc), a paired-like homeobox gene expressed in the vertebrate organizer, functions as a transcriptional repressor either by direct DNA binding to paired TAAT homeodomain sites or through recruitment by the forkhead/winged helix transcription factor Foxh1. Here, we report that Gsc is post-translationally modified by small ubiquitin-like modifier proteins (SUMO). Members of the PIAS family of proteins enhance Gsc sumoylation and this modification occurs on at least six lysine residues. Stable expression of a SUMO-defective Gsc mutant (Gsc 6Km) in MDA-MB-231 breast cancer cells results in morphological changes giving rise to cells with increased cell area. We demonstrate that Gsc 6Km can effectively repress Foxh1-mediated induction of the Mixl1 promoter, indicating that sumoylation is not required for Gsc-mediated repression of promoters where recruitment occurs through Foxh1. In contrast, Gsc 6Km exhibits a decreased ability to repress the induction of promoters to which it is directly recruited through paired-homeodomain binding sites, including its own promoter and that of the Xenopus Brachyury gene. Taken together, our data suggests that regulation of Gsc repressive activity by SUMO modification is promoter specific and may serve to differentially regulate genes that function to control cell morphology during early development and cancer.

Mesenchyme Forkhead 1 (FOXC2) plays a key role in metastasis and is associated with aggressive basal-like breast cancers.

The metastatic spread of epithelial cancer cells from the primary tumor to distant organs mimics the cell migrations that occur during embryogenesis. Using gene expression profiling, we have found that the FOXC2 transcription factor, which is involved in specifying mesenchymal cell fate during embryogenesis, is associated with the metastatic capabilities of cancer cells. FOXC2 expression is required for the ability of murine mammary carcinoma cells to metastasize to the lung, and overexpression of FOXC2 enhances the metastatic ability of mouse mammary carcinoma cells. We show that FOXC2 expression is induced in cells undergoing epithelial-mesenchymal transitions (EMTs) triggered by a number of signals, including TGF-beta1 and several EMT-inducing transcription factors, such as Snail, Twist, and Goosecoid. FOXC2 specifically promotes mesenchymal differentiation during an EMT and may serve as a key mediator to orchestrate the mesenchymal component of the EMT program. Expression of FOXC2 is significantly correlated with the highly aggressive basal-like subtype of human breast cancers. These observations indicate that FOXC2 plays a central role in promoting invasion and metastasis and that it may prove to be a highly specific molecular marker for human basal-like breast cancers.

The Spemann organizer gene, Goosecoid, promotes tumor metastasis.

The process of invasion and metastasis during tumor progression is often reminiscent of cell migration events occurring during embryonic development. We hypothesized that genes controlling cellular changes in the Spemann organizer at gastrulation might be reactivated in tumors. The Goosecoid homeobox transcription factor is a known executer of cell migration from the Spemann organizer. We found that indeed Goosecoid is overexpressed in a majority of human breast tumors. Ectopic expression of Goosecoid in human breast cells generated invasion-associated cellular changes, including an epithelial-mesenchymal transition. TGF-beta signaling, known to promote metastasis, induced Goosecoid expression in human breast cells. Moreover, Goosecoid significantly enhanced the ability of breast cancer cells to form pulmonary metastases in mice. These results demonstrate that Goosecoid promotes tumor cell malignancy and suggest that other conserved organizer genes may function similarly in human cancer.

Disruption of early proximodistal patterning and AVE formation in Apc mutants.

In the postimplantation mouse embryo, axial patterning begins with the restriction of expression of a set of genes to the distal visceral endoderm (DVE). This proximodistal (PD) axis is subsequently transformed into an anteroposterior axis as the VE migrates anteriorly to form the anterior visceral endoderm (AVE). Both Nodal and Wnt signaling pathways are involved in these events. We show here that loss of function in the adenomatous polyposis coli gene (Apc) leads to constitutive beta-catenin activity that induces a proximalization of the epiblast with the activation of a subset of posterior mesendodermal genes, and loss of ability to induce the DVE. The loss of some DVE genes such as Hex and goosecoid is rescued in chimeras where only the epiblast was wild type; however, these DVE markers were no longer restricted distally but covered the entire epiblast. Thus, the Apc gene is needed in both embryonic and extraembryonic lineages for normal PD patterning around implantation, suggesting that early restricted activation of the Wnt pathway may be important for initiating axial asymmetries. In addition, we found that nuclear beta-catenin and other molecular markers are asymmetrically expressed by 4.5 days.