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Includes supplementary tables S1, S2, S5, S6, S7 and S9

File includes supplementary figures S1 to S5

List of upregulated genes in CTC-derived brain metastases compare to other metastases (lung, bone and ovary)

Hematogenous metastasis is initiated by a subset of circulating tumor cells (CTC) shed from primary or metastatic tumors into the blood circulation. Thus, CTCs provide a unique patient biopsy resource to decipher the cellular subpopulations that initiate metastasis and their molecular properties. However, one crucial question is whether CTCs derived and expanded ex vivo from patients recapitulate human metastatic disease in an animal model. Here, we show that CTC lines established from patients with breast cancer are capable of generating metastases in mice with a pattern recapitulating most major organs from corresponding patients. Genome-wide sequencing analyses of metastatic variants identified semaphorin 4D as a regulator of tumor cell transmigration through the blood–brain barrier and MYC as a crucial regulator for the adaptation of disseminated tumor cells to the activated brain microenvironment. These data provide the direct experimental evidence of the promising role of CTCs as a prognostic factor for site-specific metastasis.Significance:Interests abound in gaining new knowledge of the physiopathology of brain metastasis. In a direct metastatic tropism analysis, we demonstrated that ex vivo–cultured CTCs from 4 patients with breast cancer showed organotropism, revealing molecular features that allow a subset of CTCs to enter and grow in the brain.This article is highlighted in the In This Issue feature, p. 1

Although the neuroinvasiveness of SARS-CoV-2 has been extensively studied, the correlation between virus infectivity and brain maturation remained unclear. Here, using human-induced pluripotent stem cells-derived three-dimensional cerebral organoids (CBOs), we present the first quantitative data for long-term kinetics of SARS-CoV-2 propagation in brain for 20 days post-infection. We showed that mature brains are more susceptible to SARS-CoV-2 than immature counterparts, evident from increased viral replication rate and higher TUNEL + cells proportion. Transcriptome profiling identified enhancement of corticogenesis and gliogenesis and indicated enrichments in translation machinery- and lipid metabolism-associated genes in mature brain, suggesting the major factors conferring the robust infectivity of SARS-CoV-2. The role of cholesterol in promoting viral replication was confirmed by the reduced number of infected cells in lipid lowering-drugs condition. Together, this study highlights that permissiveness of the brains to SARS-CoV-2 is greatly enhanced with their maturation and suggests cholesterol as a new target for suppressing viral replication.

This research aimed to compare the In Vitro growth, maturation, and gene expression in ovarian follicles collected from adult mice (6–8-week-old) between two-dimensional and three-dimensional cultures. First, we confirmed In Vitro follicle growth and maturation using adult mice with outbred characteristics and analyzed the expression of genes related to follicular development. We found that the three-dimensional culture system utilizing a Matrigel drop to create an in vivo-like ovarian microenvironment was more efficient in terms of In Vitro follicle growth, maturation, and gene expression than the two-dimensional system (non-physical environment). The in vivo-like three-dimensional culture of ovarian follicles provides new insights into the physiology and development of ovarian follicle in vivo, thereby contributing to new strategies to improve female fertility.

Glioblastoma multiforme (GBM) heterogeneity causes a greater number of deaths than any other brain tumor, despite the availability of alkylating chemotherapy. GBM stem-like cells (GSCs) contribute to GBM complexity and chemoresistance, but it remains challenging to identify and target GSCs or factors that control their activity. Here, we identified a specific GSC subset and show that activity of these cells is positively regulated by stabilization of methyl CpG binding domain 3 (MBD3) protein. MBD3 binds to CK1A and to BTRCP E3 ubiquitin ligase, triggering MBD3 degradation, suggesting that modulating this circuit could antagonize GBM recurrence. Accordingly, xenograft mice treated with the CK1A activator pyrvinium pamoate (Pyr-Pam) showed enhanced MBD3 degradation in cells expressing high levels of O(6)-methylguanine-DNA methyltransferase (MGMT) and in GSCs, overcoming temozolomide chemoresistance. Pyr-Pam blocked recruitment of MBD3 and the repressive nucleosome remodeling and deacetylase (NuRD) complex to neurogenesis-associated gene loci and increased acetyl–histone H3 activity and GSC differentiation. We conclude that CK1A/BTRCP/MBD3/NuRD signaling modulates GSC activation and malignancy, and that targeting this signaling could suppress GSC proliferation and GBM recurrence.

Summary Both 3D chromatin architecture and long non-coding RNAs (lncRNAs) play essential roles in pluripotency maintenance. However, whether lncRNAs are involved in organizing 3D chromatin structure remains largely unexplored. We identified 39 lncRNAs bound by Klf4, among which we further revealed the 5430416N02Rik promoter is a chromatin interaction hub. Knockout of the 5430416N02Rik locus reduces the proliferation rate of embryonic stem cells (ESCs). Moreover, deleting both the promoter and the gene body of 5430416N02Rik causes a more severe proliferation defect and has a more profound impact on the transcriptome than deleting the gene body alone. The reduced proliferation of the 5430416N02Rik locus knockout ESCs is mainly due to the downregulation of Mid1, the expression of which requires the inter-chromosomal interaction between Mid1 and 5430416N02Rik loci. In summary, our data demonstrated that the lncRNA 5430416N02Rik gene locus maintains the fast proliferation of ESCs by activating the expression of Mid1 through chromatin interaction., Highlights • lncRNA 5430416N02Rik participates in organizing 3D chromatin architecture • lncRNA gene 5430416N02Rik promoter is a chromatin interaction hub • Knockout of 5430416N02Rik locus reduces the proliferation rate of ESCs • Interaction between 5430416N02Rik and Mid1 loci activates Mid1 transcription, In this article, Chen and Lu and colleagues show that 5430416N02Rik, a long non-coding RNA (lncRNA) gene, is involved in organizing 3D chromatin structure in embryonic stem cells (ESCs). It is revealed that 5430416N02Rik gene serves as the chromatin interaction hub. In addition, 5430416N02Rik gene locus maintains the fast proliferation of ESCs by activating the expression of Mid1 through chromatin interaction.

Hematogenous metastasis is initiated by a subset of circulating tumor cells (CTC) shed from primary or metastatic tumors into the blood circulation. Thus, CTCs provide a unique patient biopsy resource to decipher the cellular subpopulations that initiate metastasis and their molecular properties. However, one crucial question is whether CTCs derived and expanded ex vivo from patients recapitulate human metastatic disease in an animal model. Here, we show that CTC lines established from patients with breast cancer are capable of generating metastases in mice with a pattern recapitulating most major organs from corresponding patients. Genome-wide sequencing analyses of metastatic variants identified semaphorin 4D as a regulator of tumor cell transmigration through the blood–brain barrier and MYC as a crucial regulator for the adaptation of disseminated tumor cells to the activated brain microenvironment. These data provide the direct experimental evidence of the promising role of CTCs as a prognostic factor for site-specific metastasis. Significance: Interests abound in gaining new knowledge of the physiopathology of brain metastasis. In a direct metastatic tropism analysis, we demonstrated that ex vivo–cultured CTCs from 4 patients with breast cancer showed organotropism, revealing molecular features that allow a subset of CTCs to enter and grow in the brain. This article is highlighted in the In This Issue feature, p. 1

Cancer stem cells (CSCs) are a tumor cell subpopulation that drives tumor progression and metastasis, leading to poor overall survival of patients. In colorectal cancer (CRC), hyper-activation of Wnt/β-catenin signaling by mutation of both Adenomatous polyposis coli (APC) and K-Ras increases the size of the CSC population. We previously showed that the CPD0857 inactivates Wnt/β-catenin signaling by promoting ubiquitin-dependent proteasomal degradation of β-catenin and Ras proteins, thereby decreasing proliferation and increasing apoptosis of CRC lines. CPD0857 also decreased growth and invasiveness of CRC cells harboring mutant K-Ras resistant to EGFR mAb therapy. Here, we show that CPD0857 treatment decreases proliferation and increases neuronal differentiation of neural progenitor cells (NPCs). CDP0857 effectively reduced expression of CSC markers and suppressed self-renewal capacity. CPD0857 treatment also inhibited proliferation and expression of CSC markers in D-K-Ras MT cells carrying K-Ras, APC and PI3K mutations, indicating inhibition of PI3K/AKT signaling. Moreover, CPD0857-treated xenograft mice showed regression of tumor growth and decreased numbers of CSCs in tumors. We conclude that CPD0857 could serve as the basis of a drug development strategy targeting CSCs activated through Wnt/β-catenin-Ras MAPK-PI3K/AKT signaling in CRCs.

Oncogenic K-Ras mutations in colorectal cancer (CRC) combined with APC mutations worsen CRC prognosis and lower drug effectiveness. Thus, inhibition of both Wnt/β-catenin and Ras-MAPK signaling may be a rational strategy to improve the treatment of this cancer. To identify a novel compound inhibiting both Wnt/β-catenin and Ras-MAPK signaling in CRC. We developed a two-part screening system consisting of analysis of TOP flash reporter cells and then potential toxicity effects on primary neural stem cells (NSCs). We then screened 2000 chemical compounds and tested efficacy of candidates against isogenic colon cancer cells harboring wild-type or mutant K-Ras. We employed immunohistochemistry and immunocytochemistry to determine marker signatures associated with development of disease phenotypes. We identified CPD0857, a compound that inactivates Wnt/β-catenin signaling and promotes ubiquitin-dependent proteasomal degradation of β-catenin and Ras proteins. CPD0857 effectively decreased proliferation and increased apoptosis of CRC cell lines, and overcame resistance of CRC harboring APC and K-Ras mutations to treatment with an EGFR monoclonal antibody (mAb). Moreover, CPD0857 attenuated invasiveness of highly migratory CRC cells in vitro. Accordingly, xenograft mice treated with CPD0857 showed slower tumor growth and significant decreases in both β-catenin and Ras protein expression. CPD0857 may be a potential drug for treating aggressive CRC carrying mutations that aberrantly activate Wnt/β-catenin and Ras-ERK pathways.

Hearing loss occurs with the loss of hair cells of the cochlea and subsequent degeneration of spiral ganglion neurons (SGNs). Regeneration of SGNs is a potentially promising therapeutic approach to hearing loss in addition to the use of a cochlear implant (CI), because this device stimulates SGNs directly to restore hearing bypassing the missing hair cells. The presence of SGN-neural stem cells (NSCs) has been reported in adult human and mice. These cells have the potential to become SGNs and thus represent a cellular foundation for regeneration therapies for hearing loss. Valproic acid (VPA) has been shown to influence the neural differentiation of NSCs through multiple signaling pathways involving glycogen synthase kinase3β (GSK3β). Our present study therefore aimed to modulate the neural differentiation potential of SGN-NSCs by treatment with VPA. We here report that a clinically relevant concentration of 1 mM VPA induced the differentiation of basic fibroblast growth factor (bFGF)-treated P1- and P14-SGN-NSCs into neuronal and glial cells, confirmed by neuronal marker (Tuj1 and MAP2) and glial cell marker (GFAP and S100β) detection. VPA-treated cells also promoted much longer neurite outgrowth compared to differentiated cells cultured without bFGF. The effects of VPA on the regulation of differentiation may be related to the activation of the Wnt/β-catenin signaling pathway, but not the inhibition of histone deacetylases (HDACs). We propose that VPA has the potential to convert SGN-NSCs into SGNs and thereby restore hearing when combined with a CI.

Background MMP-9-dependent proteolysis of histone H3 N-terminal tail (H3NT) is an important mechanism for activation of gene expression during osteoclast differentiation. Like other enzymes targeting their substrates within chromatin structure, MMP-9 enzymatic activity toward H3NT is tightly controlled by histone modifications such as H3K18 acetylation (H3K18ac) and H3K27 monomethylation (H3K27me1). Growing evidence indicates that DNA methylation is another epigenetic mechanism controlling osteoclastogenesis, but whether DNA methylation is also critical for regulating MMP-9-dependent H3NT proteolysis and gene expression remains unknown. Results We show here that treating RANKL-induced osteoclast progenitor (OCP) cells with the DNMT inhibitor 5-Aza-2′-deoxycytidine (5-Aza-CdR) induces CpG island hypomethylation and facilitates MMP-9 transcription. This increase in MMP-9 expression results in a significant enhancement of H3NT proteolysis and OCP cell differentiation. On the other hand, despite an increase in levels of H3K18ac, treatment with the HDAC inhibitor trichostatin A (TSA) leads to impairment of osteoclastogenic gene expression. Mechanistically, TSA treatment of OCP-induced cells stimulates H3K27ac with accompanying reduction in H3K27me1, which is a key modification to facilitate stable interaction of MMP-9 with nucleosomes for H3NT proteolysis. Moreover, hypomethylated osteoclastogenic genes in 5-Aza-CdR-treated cells remain transcriptionally inactive after TSA treatment, because H3K27 is highly acetylated and cannot be modified by G9a. Conclusions These findings clearly indicate that DNA methylation and histone modification are important mechanisms in regulating osteoclastogenic gene expression and that their inhibitors can be used as potential therapeutic tools for treating bone disorders. Electronic supplementary material The online version of this article (10.1186/s13072-019-0270-0) contains supplementary material, which is available to authorized users.

Summary Glioblastoma multiforme (GBM) is the most aggressive type of brain tumor with poor survival rate. Temozolomide (TMZ) is used as standard chemotherapy to treat GBM, but a large number of patients either respond poorly and/or develop resistance after long-term use, emphasizing the need to develop potent drugs with novel mechanisms of action. Here, using high-throughput compound screening (HTS), we found that azathioprine, an immunosuppressant, is a promising therapeutic agent to treat TMZ-resistant GBM. Through integrative genome-wide analysis and global proteomic analysis, we found that elevated lipid metabolism likely due to hyperactive EGFR/AKT/SREBP-1 signaling was inhibited by azathioprine. Azathioprine also promoted ER stress-induced apoptosis. Analysis of orthotopic xenograft models injected with patient-derived GBM cells revealed reduced tumor volume and increased apoptosis after azathioprine and TMZ co-treatment. These data indicate that azathioprine could be a powerful therapeutic option for TMZ-resistant GBM patients., Graphical abstract, Highlights • Azathioprine as a promising drug for temozolomide-resistant GBM treatment • Azathioprine inhibits aberrantly elevated lipid metabolism in GBM • Azathioprine promotes ER stress-induced apoptosis in GBM • Through orthotropic xenograft models, anti-GBM effect of azathioprine is confirmed, Biological Sciences; Cell Biology; Proteomics; Cancer Systems Biology; Cancer

Kruppel-like factor 4 (Klf4) is a zinc-finger-containing protein that plays a critical role in diverse cellular physiology. While most of these functions attribute to its role as a transcription factor, it is postulated that Klf4 may play a role other than transcriptional regulation. Here we demonstrate that Klf4 loss in neural progenitor cells (NPCs) leads to increased neurogenesis and reduced self-renewal in mice. In addition, Klf4 interacts with RNA-binding protein Staufen1 (Stau1) and RNA helicase Ddx5/17. They function together as a complex to maintain NPC self-renewal. We report that Klf4 promotes Stau1 recruitment to the 3′-untranslated region of neurogenesis-associated mRNAs, increasing Stau1-mediated mRNA decay (SMD) of these transcripts. Stau1 depletion abrogated SMD of target mRNAs and rescued neurogenesis defects in Klf4-overexpressing NPCs. Furthermore, Ddx5/17 knockdown significantly blocked Klf4-mediated mRNA degradation. Our results highlight a novel molecular mechanism underlying stability of neurogenesis-associated mRNAs controlled by the Klf4/Ddx5/17/Stau1 axis during mammalian corticogenesis., While being known as a transcription factor, Kruppel-like factor 4 (Klf4) may have other molecular functions. This study shows that Klf4 in neural progenitor cells regulate neurogenesis and self-renewal by interacting with RNA-binding protein Staufen1 and RNA helicase Ddx5/17 to control mRNA decay.

The receptor-like tyrosine kinase (Ryk), a Wnt receptor, is important for cell fate determination during corticogenesis. During neuronal differentiation, the Ryk intracellular domain (ICD) is cleaved. Cleavage of Ryk and nuclear translocation of Ryk-ICD are required for neuronal differentiation. However, the mechanism of translocation and how it regulates neuronal differentiation remain unclear. Here, we identified Smek1 and Smek2 as Ryk-ICD partners that regulate its nuclear localization and function together with Ryk-ICD in the nucleus through chromatin recruitment and gene transcription regulation. Smek1/2 double knockout mice displayed pronounced defects in the production of cortical neurons, especially interneurons, while the neural stem cell population increased. In addition, both Smek and Ryk-ICD bound to the Dlx1/2 intergenic regulator element and were involved in its transcriptional regulation. These findings demonstrate a mechanism of the Ryk signaling pathway in which Smek1/2 and Ryk-ICD work together to mediate neural cell fate during corticogenesis.

The fate of neural progenitor cells (NPCs) during corticogenesis is determined by a complex interplay of genetic or epigenetic components, but the underlying mechanism is incompletely understood. Here, we demonstrate that Suppressor of Mek null (Smek) interact with methyl-CpG–binding domain 3 (Mbd3) and the complex plays a critical role in self-renewal and neuronal differentiation of NPCs. We found that Smek promotes Mbd3 polyubiquitylation and degradation, blocking recruitment of the repressive Mbd3/nucleosome remodeling and deacetylase (NuRD) complex at the neurogenesis-associated gene loci, and, as a consequence, increasing acetyl histone H3 activity and cortical neurogenesis. Furthermore, overexpression of Mbd3 significantly blocked neuronal differentiation of NPCs, and Mbd3 depletion rescued neurogenesis defects seen in Smek1/2 knockout mice. These results reveal a novel molecular mechanism underlying Smek/Mbd3/NuRD axis-mediated control of NPCs’ self-renewal and neuronal differentiation during mammalian corticogenesis., Author summary Neural progenitor cells are self-renewing, multipotent cells that generate major neural cell types, including neurons and glia. Their fate during development of the cerebral cortex is determined by a complex interplay of genetic and epigenetic components. It has been shown that Suppressor of Mek null (Smek) proteins—which are evolutionarily conserved—play a role during the asymmetric cell division of neuroblasts in invertebrates. Methyl-CpG–binding domain 3 (Mbd3) protein, a core component of the repressive nucleosome remodeling and deacetylase (NuRD) complex, is an important epigenetic regulator that plays an essential role in mammalian development. In this study, we discovered that Smek interacts with Mbd3 and promotes its degradation via a posttranslational modification called polyubiquitylation. Degradation of Mb3, in turn, blocks recruitment of Mbd3/NuRD complex on target gene promoters, leading to an increase in neuronal differentiation during cortical development. This study not only elucidates a distinct mechanism for Smek-mediated neuronal differentiation but also identifies Smek as a negative regulator of the Mbd3 protein during cortical brain development.

Aims The activation of Wnt/β-catenin signaling pathway plays an important role in hair follicle morphogenesis by stimulating bulge stem cells. This study was to obtain the activator of Wnt/β-catenin signaling pathway from natural products and to determine whether this activator can induce anagen hair growth in mice. Main methods To identify materials that activate Wnt/β-catenin signaling pathway, 800 natural product extracts were screened using pTOPFlash assay and neural progenitor cell (NPC) differentiation assay. A selected extract was further tested for its effects on alkaline phosphatase (ALP) activity in human immortalized dermal papilla cell (iDPC) and the proliferation in iDPC and immortalized rat vibrissa DPC (RvDP). Finally, hair growth-promoting effects were evaluated in the dorsal skin of C57BL/6 mice. Key findings Aconiti Ciliare Tuber (ACT) extract was one of the most active materials in both pTOPFlash and NPC differentiation assays. It promoted the differentiation of NPC cells even under proliferation-stimulating conditions (basic fibroblast growth factor: bFGF). It also increased ALP activity and proliferation of iDPC in dose-dependent manners, and it stimulated the induction of the anagen hair growth in C57BL/6 mice. These results suggest that ACT extract activates the Wnt/β-catenin signaling pathway by enhancing β-catenin transcription and has the potential to promote the induction of hair growth via activation of the stem cell activity of the dermal papilla cells. Significance This is the first report indicating benefits of ACT extract in hair loss prevention by triggering the activation of Wnt/β-catenin signaling pathway and induction of the anagen hair growth in mice.

Sur8/Shoc2 is a scaffold protein that regulates the Ras-extracellular signal-regulated kinase (ERK) pathway. However, the roles of Sur8 in cellular physiologies are poorly understood. In this study, Sur8 was severely repressed in the course of neural progenitor cell (NPC) differentiation in the cerebral cortex of developing rat embryos. Similarly, Sur8 was also critically reduced in cultured NPCs, which were induced differentiation by removal of basic fibroblast growth factor (bFGF). Sur8 regulation occurs at the protein level rather than at the mRNA level as revealed by both in situ hybridization and reverse transcriptase polymerase chain reaction analyses. The role of Sur8 in NPC differentiation was confirmed by lentivirus-mediated Sur8 knockdown, which resulted in increased differentiation, whereas exogenous expression of Sur8 inhibited differentiation. Contrastingly, NPC proliferation was promoted by overexpression, but was suppressed by Sur8 knockdown. The role of Sur8 as an antidifferentiation factor in the developing rat brain was confirmed by an ex vivo embryo culture system combined with the lentivirus-mediated Sur8 knockdown. The numbers and sizes of neurospheres were reduced, but neuronal outgrowth was enhanced by the Sur8 knockdown. The Ras-ERK pathway is involved in Sur8-mediated regulations of differentiation, as the treatment of ERK kinase (MEK) inhibitors blocks the effects of Sur8. The regulations of NPCs' differentiation and proliferation by the Ras-ERK pathway were also shown by the rescues of the effects of bFGF depletion, neuronal differentiation, and antiproliferation by epidermal growth factor. In summary, Sur8 is an antidifferentiation factor that stimulates proliferation for maintenance of self-renewal in NPCs via modulation of the Ras-ERK pathway.

During tumor progression, tumor cells invade the primary tumor microenvironment and intravasate into blood vessels, where they are referred to as circulating tumor cells (CTCs). These CTCs disseminate to other organs, and a subset of these cells will form metastasis. The growing interest for CTCs is confronted with the difficulty associated with their isolation and scarcity of material. To address this challenge, our established CTC lines from breast cancer patients offered a unique opportunity to investigate the signal pathways involved in the metastatic cascade. We assessed the metastatic potential of breast cancer patient-derived CTC lines by injecting them directly into the left ventricle of the heart in NSG mice. CTC lines are capable of generating metastases in most organs that are listed as common sites of metastases from breast cancer, such as the brain, lungs, bones and ovaries. Two patient-derived CTC lines harbored a high metastatic potential (over 80% of mice had metastases after 3 months) with generation of simultaneous metastases in the bones, lungs and ovaries. These mice remained brain-metastases free for up to 8 months. However, in 2 patient-derived CTC lines we demonstrated the brain as the preferential site of metastasis despite their low metastatic potential. Interestingly, in our mouse model, CTC lines recapitulated human cancer metastatic progression, as shown by the clinical data collected from patients where CTC lines were generated. We further investigated genetic and epigenetic determinants that regulate the organotropism of CTCs. To address this we isolated metastatic variants with a preferential tropism for the brain, lungs and bones. Investigating the gene expression signatures of metastatic variants lead us to identify the semaphorin 4D as a possible regulator of the brain metastasis. High SEMA4D expression at the primary site correlated with decreased brain metastasis-free survival in the breast cancer patients. Amplification of the chromosome 9 where the SEMA4D gene resides was also found in CTC-derived brain metastases. The SEMA4D overexpression promoted the ability of CTCs to transmigrate the blood-brain-barrier in vitro. We simultaneously investigated expression changes in stromal cells in organ-specific metastases, and among the pathways with the highest enrichment scores we found TNF and IFN signaling in the brain metastatic microenvironment. Next we used ATAC-seq to assay chromatin accessibility and identified potential regulatory factors mediating the brain metastasis formation. These data provides the evidence for the promising role of CTCs as an early prognostic factor for metastasis. Additionally, we expect to develop novel organ tropism associated markers, which can be considered for potential therapeutic targets in breast cancer. Citation Format: Remi Klotz, Amal Thomas, Sara Restrepo-Vassalli, Oihana Iriondo, Jane Han, Teng Teng, Matthew MacKay, Alan Wang, Negeen Izadian, Byoung-San Moon, Aditya Bardia, James Hicks, Andrew Smith, Min Yu. Dissecting mechanisms of breast cancer metastasis through patient-derived circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3023.

Both the Wnt/β-catenin and Ras pathways are aberrantly activated in most human colorectal cancers (CRCs) and interact cooperatively in tumor promotion. Inhibition of these signaling may therefore be an ideal strategy for treating CRC. We identified KY1220, a compound that destabilizes both β-catenin and Ras, via targeting the Wnt/β-catenin pathway, and synthesized its derivative KYA1797K. KYA1797K bound directly to the regulators of G-protein signaling domain of axin, initiating β-catenin and Ras degradation through enhancement of the β-catenin destruction complex activating GSK3β. KYA1797K effectively suppressed the growth of CRCs harboring APC and KRAS mutations, as shown by various in vitro studies and by in vivo studies using xenograft and transgenic mouse models of tumors induced by APC and KRAS mutations. Destabilization of both β-catenin and Ras via targeting axin is a potential therapeutic strategy for treatment of CRC and other type cancers activated Wnt/β-catenin and Ras pathways.

BACKGROUND Adenomatous polyposis coli (APC) loss-of-function mutations and K-Ras gain-of-function mutations are common abnormalities that occur during the initiation and intermediate adenoma stages of colorectal tumorigenesis, respectively. However, little is known about the role these mutations play in cancer stem cells (CSCs) associated with colorectal cancer (CRC) tumorigenesis. METHODS We analyzed tissue from CRC patients (n = 49) to determine whether K-Ras mutations contributed to CSC activation during colorectal tumorigenesis. DLD-1-K-Ras-WT and DLD-1-K-Ras-MT cells were cultured and evaluated for their ability to differentiate, form spheroids in vitro, and form tumors in vivo. Interaction between APC and K-Ras mutations in colorectal tumorigenesis was evaluated using APC (Min/+)/K-Ras (LA2) mice and DLD-1-K-Ras-WT and DLD-1-K-Ras-MT cell xenografts. (n = 4) Group differences were determined by Student t test. All statistical tests were two-sided. RESULTS The sphere-forming capability of DLD-1-K-Ras-MT cells was statistically significantly higher than that of DLD-1-K-Ras-WT cells (DLD-1-K-Ras-MT mean = 86.661 pixel, 95% confidence interval [CI] = 81.701 to 91.621 pixel; DLD-1-K-Ras-WT mean = 42.367 pixel, 95% CI = 36.467 to 48.267 pixel; P = .003). Moreover, both the size and weight of tumors from DLD-1-K-Ras-MT xenografts were markedly increased compared with tumors from DLD-1-K-Ras-WT cells. Expression of the CSC markers CD44, CD133, and CD166 was induced in intestinal tumors from APC (Min/+)/K-Ras (LA2)mice, but not K-Ras (LA2) mice, indicating that APC mutation is required for CSC activation by oncogenic K-Ras mutation. CONCLUSIONS K-Ras mutation activates CSCs, contributing to colorectal tumorigenesis and metastasis in CRC cells harboring APC mutations. Initial activation of β-catenin by APC loss and further enhancement through K-Ras mutation induces CD44, CD133, and CD166 expression.

Neural progenitor cells (NPCs) or neural stem cells are important tools for investigating central nervous system (CNS) development. NPCs can be used in therapeutic strategies and for characterizing differentiation mechanisms. Here, we describe methods for isolating and culturing embryonic NPCs.

Sphingosine-1-phosphate (S1P) signaling regulates lymphocyte egress from lymphoid organs into systemic circulation. Sphingosine phosphate receptor 1 (S1P1) agonist, FTY-720 (Gilenya™) arrests immune trafficking and prevents multiple sclerosis (MS) relapses. However, alternative mechanisms of S1P-S1P1 signaling have been reported. Phosphoproteomic analysis of MS brain lesions revealed S1P1 phosphorylation on S351, a residue crucial for receptor internalization. Mutant mice harboring a S1pr1 gene encoding phosphorylation-deficient receptors [S1P1(S5A)] developed severe experimental autoimmune encephalomyelitis (EAE) due to T helper (TH) 17-mediated autoimmunity in the peripheral immune and nervous system. S1P1 directly activated Janus-like kinase–signal transducer and activator of transcription 3 (JAK-STAT3) pathway via interleukin 6 (IL-6). Impaired S1P1 phosphorylation enhances TH17 polarization and exacerbates autoimmune neuroinflammation. These mechanisms may be pathogenic in MS.

Neural progenitor cells (NPCs) or neural stem cells are important tools for investigating central nervous system (CNS) development. NPCs can be used in therapeutic strategies and for characterizing differentiation mechanisms. Here, we describe methods for isolating and culturing embryonic NPCs.

Recent evidence suggests that many endogenous circular RNAs (circRNAs) may play roles in biological processes. However, the expression patterns and functions of circRNAs in human diseases are not well understood. Computationally identifying circRNAs from total RNA-seq data is a primary step in studying their expression pattern and biological roles. In this work, we have developed a computational pipeline named UROBORUS to detect circRNAs in total RNA-seq data. By applying UROBORUS to RNA-seq data from 46 gliomas and normal brain samples, we detected thousands of circRNAs supported by at least two read counts, followed by successful experimental validation on 24 circRNAs from the randomly selected 27 circRNAs. UROBORUS is an efficient tool that can detect circRNAs with low expression levels in total RNA-seq without RNase R treatment. The circRNAs expression profiling revealed more than 476 circular RNAs differentially expressed in control brain tissues and gliomas. Together with parental gene expression, we found that circRNA and its parental gene have diversified expression patterns in gliomas and control brain tissues. This study establishes an efficient and sensitive approach for predicting circRNAs using total RNA-seq data. The UROBORUS pipeline can be accessed freely for non-commercial purposes at http://uroborus.openbioinformatics.org/.

Background Valproic acid (VPA), a commonly used mood stabilizer that promotes neuronal differentiation, regulates multiple signaling pathways involving extracellular signal-regulated kinase (ERK) and glycogen synthase kinase3β (GSK3β). However, the mechanism by which VPA promotes differentiation is not understood. Results We report here that 1 mM VPA simultaneously induces differentiation and reduces proliferation of basic fibroblast growth factor (bFGF)-treated embryonic day 14 (E14) rat cerebral cortex neural progenitor cells (NPCs). The effects of VPA on the regulation of differentiation and inhibition of proliferation occur via the ERK-p21Cip/WAF1 pathway. These effects, however, are not mediated by the pathway involving the epidermal growth factor receptor (EGFR) but via the pathway which stabilizes Ras through β-catenin signaling. Stimulation of differentiation and inhibition of proliferation in NPCs by VPA occur independently and the β-catenin-Ras-ERK-p21Cip/WAF1 pathway is involved in both processes. The independent regulation of differentiation and proliferation in NPCs by VPA was also demonstrated in vivo in the cerebral cortex of developing rat embryos. Conclusion We propose that this mechanism of VPA action may contribute to an explanation of its anti-tumor and neuroprotective effects, as well as elucidate its role in the independent regulation of differentiation and inhibition of proliferation in the cerebral cortex of developing rat embryos.

Adenomatous polyposis coli (APC) loss-of-function mutations and K-Ras gain-of-function mutations are common abnormalities that occur during the initiation and intermediate adenoma stages of colorectal tumorigenesis, respectively. However, little is known about the role of these mutations in the regulation of cancer stem cells (CSCs) associated with colorectal cancer (CRC) tumorigenesis. We analyzed tissue from CRC patients (n = 49) and observed an association between K-Ras mutations and CSC activation during colorectal tumorigenesis. The oncogenic K-Ras mutations enhanced stemness of CRC cells carrying an APC mutation, as shown by comparisons of sphere formation, transforming potential, and differentiation in vitro and tumor-forming capacity in vivo between isogenic APC mutated CRC cells harboring either wild-type or mutant K-Ras. Moreover, the activation of CSC by K-Ras mutations in CRC required an additional APC mutation, as revealed by the induction of CD44, CD133, and CD166 in intestinal tumor tissues of APCMin/+/K-RasLA2 double-mutant mice, but not in K-RasLA2 mice. Overall, the progression and metastasis of CRC induced by K-Ras mutation occurs by initial activation of CSC by APC loss and further activation of Wnt/β-catenin signaling and subsequent activation of Ras-ERK signaling. Citation Format: Byoung-San Moon, Kug Hwa Lee, Kyounghwa Koo, Kang-Yell Choi. Activation of cancer stem cells by oncogenic K-Ras requires activated Wnt/β-catenin signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2330. doi:10.1158/1538-7445.AM2015-2330

Bone morphogenic protein 4 (BMP4), a member of the TGF-beta superfamily, induced neural differentiation of neural stem cells (NSCs) grown in a medium containing basic fibroblast growth factor (bFGF). The Ras protein level and the activities of the downstream ERKs were increased by transfection of BMP4 or treatment with recombinant BMP4. The effects of BMP4, including activation of the Ras-ERK pathway and induction of the neuron marker beta-tubulin type III (Tuj1), were blocked by co-treatment of the BMP4 antagonist, noggin. The roles of the Ras-ERK pathway in neuronal differentiation by BMP4 were revealed by measuring the effect of the ERK pathway inhibition by dominant negative Ras or PD98059, the MEK specific inhibitor. BMP4 is a transcriptional target of Wnt/beta-catenin signaling, and both the mRNA and protein levels of BMP4 were increased by treatment of valproic acid (VPA), a chemical inhibitor of glycogen synthase kinase 3beta (GSK3beta) activating the Wnt/beta-catenin pathway. The BMP4- mimicking effects of VPA, activation of the Ras-ERK pathway and induction of Tuj1, also were blocked by noggin. These results indicate the potential therapeutic usage of VPA as a replacement for BMP4.