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Breast cancer has a high risk of recurrence and distant metastasis after remission. Controlling distant metastasis is important for reducing breast cancer mortality, but accomplishing this goal remains elusive. In this study, we investigated the molecular pathways underlying metastasis using cells that mimic the breast cancer distant metastasis process. HCC1143 breast cancer cells were cultured under two-dimensional (2D)-adherent, tumor sphere (TS), and reattached (ReA) culture conditions to mimic primary tumors, circulating tumor cells, and metastasized tumors, respectively. ReA cells demonstrated increased TS formation and enhanced invasion capacity compared to the original 2D-cultured parental cells. In addition, ReA cells had a higher frequency of ESA+CD44+CD24− population, which represents a stem-cell-like cell population. RNA sequencing identified the cholesterol synthesis pathway as one of the most significantly increased pathways in TS and ReA cells compared to parental cells, which was verified by measuring intracellular cholesterol levels. Furthermore, the pharmacological inhibition of the cholesterol synthesis pathway decreased the ability of cancer cells to form TSs and invade. Our results suggest that the cholesterol synthesis pathway plays an important role in the distant metastasis of breast cancer cells by augmenting TS formation and invasion capacity.

Sox2 is a core transcription factor in embryonic stem cells (ESCs), and O-GlcNAcylation is a type of post-translational modification of nuclear-cytoplasmic proteins. Although both factors play important roles in the maintenance and differentiation of ESCs and the serine 248 (S248) and threonine 258 (T258) residues of Sox2 are modified by O-GlcNAcylation, the function of Sox2 O-GlcNAcylation is unclear. Here, we show that O-GlcNAcylation of Sox2 at T258 regulates mouse ESC self-renewal and early cell fate. ESCs in which wild-type Sox2 was replaced with the Sox2 T258A mutant exhibited reduced self-renewal, whereas ESCs with the Sox2 S248A point mutation did not. ESCs with the Sox2 T258A mutation heterologously introduced using the CRISPR/Cas9 system, designated E14-Sox2TA/WT, also exhibited reduced self-renewal. RNA sequencing analysis under self-renewal conditions showed that upregulated expression of early differentiation genes, rather than a downregulated expression of self-renewal genes, was responsible for the reduced self-renewal of E14-Sox2TA/WT cells. There was a significant decrease in ectodermal tissue and a marked increase in cartilage tissue in E14-Sox2TA/WT-derived teratomas compared with normal E14 ESC-derived teratomas. RNA sequencing of teratomas revealed that genes related to brain development had generally downregulated expression in the E14-Sox2TA/WT-derived teratomas. Our findings using the Sox2 T258A mutant suggest that Sox2 T258 O-GlcNAc has a positive effect on ESC self-renewal and plays an important role in the proper development of ectodermal lineage cells. Overall, our study directly links O-GlcNAcylation and early cell fate decisions., Stem cell development: hold the sugar Cells that can grow into any type of cell, called embryonic stem cells (ESCs), are signaled to stay in stem cell mode (maintain stemness) by addition of a single sugar molecule to Sox2, a regulatory protein coded for by a developmental gene. Sugar modification of Sox2 was known to be involved in maintaining stemness and sometimes implicated in cancer, but the mechanism was poorly understood. When Hyonchol Jang at the National Cancer Center in South Korea and co-workers prevented sugar modification of Sox2 by changing an amino acid at the sugar-binding site, ESCs showed reduced self-renewal. Rather than repressing genes related to stemness, the modification failed to repress developmental genes, permitting cells to grow into other cell types. These results illuminate both the role of Sox2 in cancer and the importance of sugar modification in stemness.

Glioblastoma (GBM) is the most lethal primary brain tumor with few treatment options. The survival of glioma-initiating cells (GICs) is one of the major factors contributing to treatment failure. GICs frequently produce and respond to their own growth factors that support cell proliferation and survival. In this study, we aimed to identify critical autocrine factors mediating GIC survival and to evaluate the anti-GBM effect of antagonizing these factors. Proteomic analysis was performed using conditioned media from two different patient-derived GBM tumor spheres under a growth factor-depleted status. Then, the antitumor effects of inhibiting an identified autocrine factor were evaluated by bioinformatic analysis and molecular validation. Proteins secreted by sphere-forming GICs promote cell proliferation/survival and detoxify reactive oxygen species (ROS). Among these proteins, we focused on midkine (MDK) as a clinically significant and pathologically relevant autocrine factor. Antagonizing MDK reduced the survival of GBM tumor spheres through the promotion of cell cycle arrest and the consequent apoptotic cell death caused by oxidative stress-induced DNA damage. We also identified PCBP4, a novel molecular predictor of resistance to anti-MDK treatment. Collectively, our results indicate that MDK inhibition is an important therapeutic option by suppressing GIC survival through the induction of ROS-mediated cell cycle arrest and apoptosis., Brain cancer: a key growth factor Targeting the growth factor midkine (MDK) may offer improved treatment for glioblastoma, the most lethal brain cancer. In glioblastoma, the tumor-initiating cells produce their own growth factors, encouraging themselves to keep multiplying, and making glioblastoma very difficult to treat. Do-Hyun Nam and Hyun Ju Kang at the Samsung Medical Center, Seoul, South Korea and coworkers screened the growth factors produced by two glioblastoma samples, then focused on a single cancer-associated factor, MDK. Antagonizing MDK slowed tumor cell growth, and further investigation showed that suppressing MDK restored the susceptibility of tumor cells to DNA damage and the mechanisms that weed out damaged cells. Noting that treatment efficacy varied between tumor samples, the researchers identified a biomarker for sensitivity to MDK treatment. These results point the way to new treatments for this particularly deadly cancer.

Simple Summary Real-time ex vivo drug testing tailors individual therapeutics based on predicted drug responses. Most technologies to date rely on conventional drug screening that provides low confidence data. Here, we present high-content analysis-based drug testing of glioblastoma patients to identify the right glioblastoma patients for a given drug. This generates multi-parameter biomarker and phenotype readouts providing a better reliability of the assay. Additionally, we showed a high-content drug repurposing screen and defined a new c-Met-inhibiting function of the CDK4/6 inhibitor Abemaciclib. Large-scale high throughput screening results demonstrate that Abemaciclib sensitivity in glioblastoma patients is highly correlated with the c-Met inhibitors sensitivity, further supporting the accuracy of the platform and important new clinical implications regarding multiple functions of Abemaciclib. Abstract (1) Background: Recent advances in precision oncology research rely on indicating specific genetic alterations associated with treatment sensitivity. Developing ex vivo systems to identify cancer patients who will respond to a specific drug remains important. (2) Methods: cells from 12 patients with glioblastoma were isolated, cultured, and subjected to high-content screening. Multi-parameter analyses assessed the c-Met level, cell viability, apoptosis, cell motility, and migration. A drug repurposing screen and large-scale drug sensitivity screening data across 59 cancer cell lines and patient-derived cells were obtained from 125 glioblastoma samples. (3) Results: High-content analysis of patient-derived cells provided robust and accurate drug responses to c-Met-targeted agents. Only the cells of one glioblastoma patient (PDC6) showed elevated c-Met level and high susceptibility to the c-Met inhibitors. Multi-parameter image analysis also reflected a decreased c-Met expression and reduced cell growth and motility by a c-Met-targeting antibody. In addition, a drug repurposing screen identified Abemaciclib as a distinct CDK4/6 inhibitor with a potent c-Met-inhibitory function. Consistent with this, we present large-scale drug sensitivity screening data showing that the Abemaciclib response correlates with the response to c-Met inhibitors. (4) Conclusions: Our study provides a new insight into high-content screening platforms supporting drug sensitivity prediction and novel therapeutics screening.

There are large uncertainties in the single-scattering (i.e., morphologies) and microphysical (i.e., concentrations) properties of ice particles whose size are less than ~100 µm. Insufficient resolutions of the most advanced cloud probes (e.g., cloud particle imager) cannot resolve the micrometer-scale morphologies of small ice particles. Further, the shattering of large ice particles on probes’ inlets or tips causes uncertainties in the measurement of the concentrations of small ice particles. These uncertainties have large impacts on the single-scattering and microphysical properties of small ice particles that are utilized to quantify the bulk radiative properties of cirrus. In this study, the impacts of uncertainties in the morphologies and concentrations of small ice particles on the bulk radiative properties of tropical cirrus were calculated using measurements acquired during the Tropical Warm Pool-International Cloud Experiment. Five different models (i.e., budding Buckyball, Chebyshev particle, droxtal, Gaussian random sphere, and sphere) that represent the shapes of small ice particles were used to calculate the single-scattering properties. The bulk radiative properties, average phase-function (P11¯), and average asymmetry parameter (g¯) were computed by combining the measured size/habit distributions and the calculated single-scattering properties of ice particles. The impacts of the selection of varying morphologies of small particles on the bulk radiative properties were quantified. For these calculations, the possible range of the concentrations of small ice particles which depend on the degree of shattered large particles were also used. The impacts of varying the single-scattering properties of small ice particles on the bulk radiative properties were the largest in the upper parts of cirrus (T < −60 °C), while they were the smallest in the lower parts of cirrus (−45 < T < −30 °C). The impacts of uncertainties in the concentrations of small ice particles on the bulk radiative properties were largest in the lower parts of cirrus (−45 < T < −30 °C), whereas they were smallest in the upper parts of cirrus (T < −60 °C). The effect of shattering was maximum in the lower parts of cirrus, whilst it was minimum in the upper parts of cirrus. The combined impacts of uncertainties in the single-scattering (i.e., morphologies) and microphysical (i.e., concentrations) properties of small ice particles revealed variations of up to 11.2% (127.1%; 67.3%) of the integrated intensity in the forward (sideward; backward) angles in P11¯ and a corresponding change in g¯ by up to 12.61%.

Glioblastoma is a highly aggressive and lethal brain tumor, with limited treatment options. Abnormal activation of the neddylation pathway is observed in glioblastoma, and the NEDD8-activating enzyme (NAE) inhibitor, MLN4924, was previously shown to be effective in glioblastoma cell line models. However, its effect has not been tested in patient-derived glioblastoma stem cells. We first analyzed public data to determine whether NEDD8 pathway proteins are important in glioblastoma development and patient survival. NAE1 and UBA3 levels increased in glioblastoma patients, high NEDD8 levels were associated with poor clinical outcomes. Immunohistochemistry results also supported this result. The effects of MLN4924 were evaluated in 4 glioblastoma cell lines and 15 patient-derived glioblastoma stem cells using high content analysis. Glioblastoma cell lines and patient-derived stem cells were highly susceptible to MLN4924, while normal human astrocytes were resistant. In addition, there were various responses in 15 patient-derived glioblastoma stem cells upon MLN4924 treatment. Genomic analyses indicated that MLN4924 sensitive cells exhibited enrichment of Extracellular Signal Regulated Kinase (ERK) and Protein kinase B (AKT, also known as PKB) signaling. We verified that MLN4924 inhibits ERK and AKT phosphorylation in MLN4924 sensitive cells. Our findings suggest that patient-derived glioblastoma stem cells in the context of ERK and AKT activation are sensitive and highly regulated by neddylation inhibition.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Simple Summary Octamer-binding transcription factor 4 (OCT4) plays an important role in early embryonic development, but is rarely expressed in adults. However, in many cancer cells, this gene is re-expressed, making the cancer malignant. This present study revealed that inhibiting OCT4 transcriptional activity induces cancer cell differentiation and growth retardation. Specifically, when the phosphorylation of OCT4 serine 236 increases by interfering with the binding of protein phosphatase 1 (PP1) to OCT4, OCT4 loses its transcriptional activity and cancer cells differentiate. Therefore, this study presents the basis for the development of protein-protein interaction inhibitors that inhibit the binding of OCT4 and PP1 for cancer treatment. Abstract Octamer-binding transcription factor 4 (Oct4) plays an important role in maintaining pluripotency in embryonic stem cells and is closely related to the malignancies of various cancers. Although posttranslational modifications of Oct4 have been widely studied, most of these have not yet been fully characterized, especially in cancer. In this study, we investigated the role of phosphorylation of serine 236 of OCT4 [OCT4 (S236)] in human germ cell tumors (GCTs). OCT4 was phosphorylated at S236 in a cell cycle-dependent manner in a patient sample and GCT cell lines. The substitution of endogenous OCT4 by a mimic of phosphorylated OCT4 with a serine-to-aspartate mutation at S236 (S236D) resulted in tumor cell differentiation, growth retardation, and inhibition of tumor sphere formation. GCT cells expressing OCT4 S236D instead of endogenous OCT4 were similar to cells with OCT4 depletion at the mRNA transcript level as well as in the phenotype. OCT4 S236D also induced tumor cell differentiation and growth retardation in mouse xenograft experiments. Inhibition of protein phosphatase 1 by chemicals or short hairpin RNAs increased phosphorylation at OCT4 (S236) and resulted in the differentiation of GCTs. These results reveal the role of OCT4 (S236) phosphorylation in GCTs and suggest a new strategy for suppressing OCT4 in cancer.

Testing new ways to identify untapped opportunities for glioblastoma therapies remains highly significant. Amplification and overexpression of MDM2 gene is frequent in glioblastoma and disrupting the MDM2−p53 interaction is a promising strategy to treat the cancer. RG7112 is the first-in class inhibitor and recently discovered AMG232 is the most potent MDM2 inhibitor known to date. Here, we compared the effects of these two clinical MDM2 inhibitors in six glioblastoma cell lines and ten patient-derived glioblastoma stem cells. Targeted sequencing of the TP53, MDM2 genes and whole transcriptome analysis were conducted to verify genetic status associated with sensitivity and resistance to the drugs. Although TP53 wild-type glioblastoma cell lines are similarly sensitive to AMG232 and RG7112, we found that four TP53 wild-type out of ten patient-derived glioblastoma cells are much more sensitive to AMG232 than RG7112 (average IC50 of 76 nM vs. 720 nM). Among these, 464T stem cells containing MDM2 gene amplification were most sensitive to AMG232 with IC50 of 5.3 nM. Moreover, AMG232 exhibited higher selectivity against p53 wild-type cells over p53 mutant stem cells compared to RG7112 (average selectivity of 512-fold vs. 16.5-fold). Importantly, we also found that AMG232 is highly efficacious in three-dimensional (3D) tumor spheroids growth and effectively inhibits the stemness-related factors, Nestin and ZEB1. Our data provide new evidence that glioblastoma stem cells have high susceptibility to AMG232 suggesting the potential clinical implications of MDM2 inhibition for glioblastoma treatment. These will facilitate additional preclinical and clinical studies evaluating MDM2 inhibitors in glioblastoma and direct further efforts towards developing better MDM2-targeted therapeutics.

Glioblastoma (GBM) is the most lethal brain cancer with profound genomic alterations. While the bona fide tumor suppressor genes such as PTEN, NF1, and TP53 have high frequency of inactivating mutations, there may be the genes with GBM-suppressive roles for which genomic mutation is not a primary cause for inactivation. To identify such genes, we employed in vivo RNAi screening approach using the patient-derived GBM xenograft models. We found that Nemo-Like Kinase (NLK) negatively regulates mesenchymal activities, a characteristic of aggressive GBM, in part via inhibition of WNT/β-catenin signaling. Consistent with this, we found that NLK expression is especially low in a subset of GBMs that harbors high WNT/mesenchymal activities. Restoration of NLK inhibited WNT and mesenchymal activities, decreased clonogenic growth and survival, and impeded tumor growth in vivo. These data unravel a tumor suppressive role of NLK and support the feasibility of combining oncogenomics with in vivo RNAi screen.

GBM is the most aggressive human primary brain tumor and leads to poor clinical outcomes. According to previous studies, exogenous factors secreted from primary brain tumor cells can change the growth pattern of normal cells. We analyzed the ingredients of cell culture media from two patient glioblastoma cells which can grow without growth factors. As a result, we selected a significant factor for tumor cell growth which is called cytokine A, the Heparin-binding growth factor. Because of the cell viability maintenance ability of cytokine A, we tried to investigate detailed mechanism of cytokine A and applied the result to the combination therapy. Cytokine A is critical for tumorigenesis of GBM by modulating DNA damage signaling and cell cycle through ROS. For that reason, cytokine A is effective therapeutic target and can be used in combination therapy.

GBM is the most aggressive human primary brain tumor and leads to poor clinical outcomes. According to previous studies, exogenous factors secreted from primary brain tumor cells can change the growth pattern of normal cells. We analyzed the ingredients of cell culture media from two patient glioblastoma cells which can grow without growth factors. As a result, we selected a significant factor for tumor cell growth which is called cytokine A, the Heparin-binding growth factor. Because of the cell viability maintenance ability of cytokine A, we tried to investigate detailed mechanism of cytokine A and applied the result to the combination therapy. Cytokine A is critical for tumorigenesis of GBM by modulating DNA damage signaling and cell cycle through ROS. For that reason, cytokine A is effective therapeutic target and can be used in combination therapy. Citation Format: Suji Han, Kayoung Shin, Kyoungmin Lee, Sungsoo Kim, Hyunju Kang, Jin-Ku Lee, Hyun Nam, Kyeung Min Joo, Do-Hyun Nam. Patient GBM cell originated secretome analysis identifies cytokine A, as a potent therapeutic target [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 2521.

We have developed and tested a new ultrasonic technique for rapid, remote inspection of honeycomb and similar sandwich composites based on imaging of local resonance behavior. A single pristine honeycomb cell can be approximately modeled as a hexagonal plate with appropriate boundary conditions on its six sides. With this in mind, we excite the entire honeycomb panel continuously with a single, steady tone at the resonance frequency of the single pristine cell using a fixed-position transducer. We then measure and visualize the full-field amplitude and phase response over the inspection area using a scanning laser Doppler vibrometer. We applied the technique to two imperfect aluminum honeycomb panels. By imaging the magnitude of the response at each pixel, we found that one can simultaneously visualize the high-amplitude response of the pristine cells, the lower-amplitude response of the out-of-spec cells, the near-zero response near the cell boundaries, and the non-zero response in the damaged cell walls.

Most bone grafts performed today utilize autograft, allograft, and alloplast with moderate clinical success1-3). Autografts have the advantage of optimal biologic incorporation, histocompatibility and little chance of disease transmission. But it has lack of availability in clinical use, and the harvesting of graft tissue may cause patient morbidity. In contrast, allografts get much wider availability and little patient morbidity associated with graft procurement. However, problems with unreliable graft incorporation, immune response, and possible disease transmission represent clear drawbacks to their use. While several synthetic graft materials have also been introduced, most alloplastic materials function primarily as a biocompatible defect filler4). Several attempts have been made to resolve these problem use of biodegradable polymers and combination of ceramics with bioactive polymers such as collagen and polylactides5-7). Chitosan is a biodegradable cationic polysaccharide composed of N-acetylglucosamine residues which is known to accelerate wound healing and bone formation8). Many previous reports corroborated enhanced wound healing912) and hemostatic effect13-17) of chitosan. It is hypothesized that the major path for chitosan breakage down in vivo is through lysozyme, which acts slowly to depolymerize the polysaccharide18). The biodegradation rate of the polymer is determined by the amount of residual acetyl content, a parameter that can easily be varied8). One possible approach toward addressing the respective problems inherent in autograft and allograft material is employing tissue engineering which is actively researched today. Tissue engineering is a science in which material properties of synthetic compounds are manipulated to enable delivery of an aggregate of dissociated cells into host in a manner that result in the formation of new tissue. Thus the major goal of tissue engineering is in vitro construction of transplantable vital tissue19).