Kemi Cui

Aurora-B Mediated ATM Serine 1403 Phosphorylation Is Required For Mitotic ATM Activation and the Spindle Checkpoint

The ATM kinase plays a critical role in the maintenance of genetic stability. ATM is activated in response to DNA damage and is essential for cell-cycle checkpoints. Here, we report that ATM is activated in mitosis in the absence of DNA damage. We demonstrate that mitotic ATM activation is dependent on the Aurora-B kinase and that Aurora-B phosphorylates ATM on serine 1403. This phosphorylation event is required for mitotic ATM activation. Further, we show that loss of ATM function results in shortened mitotic timing and a defective spindle checkpoint, and that abrogation of ATM Ser1403 phosphorylation leads to this spindle checkpoint defect. We also demonstrate that mitotically activated ATM phosphorylates Bub1, a critical kinetochore protein, on Ser314. ATM-mediated Bub1 Ser314 phosphorylation is required for Bub1 activity and is essential for the activation of the spindle checkpoint. Collectively, our data highlight mechanisms of a critical function of ATM in mitosis.

A quantitative study of factors affecting in vivo bioluminescence imaging

In vivo bioluminescence imaging (BLI) has the advantages of high sensitivity and low background. By counting the number of photons emitted from a specimen, BLI can quantify biological events such as tumour growth, gene expression and drug response. The intensities and kinetics of the BL signal are affected by many factors and may confound the quantitative results acquired from consecutive imaging sessions or different specimens. We used three different mouse models of tumours to examine whether anaesthetics, positioning and tumour growth may affect the consistency of the BL signal. The results showed that BLI signal could be affected by different anaesthetics and repetitive positioning. Using the same anaesthetics produced consistent peak times, while other factors were held constant. However, as the tumours grew the peak times shifted and the time course of BL signals had different shapes, depending on the positioning of the mice. The data indicate that a carefully designed BLI experiment is required to generate optimal and consistent results.

In Vivo Visualization and Characterization of Epithelial–Mesenchymal Transition in Breast Tumors

The activation of the epithelial-to-mesenchymal transition (EMT) program is a critical step in cancer progression and metastasis, but visualization of this process at the single-cell level, especially in vivo, remains challenging. We established an in vivo approach to track the fate of tumor cells based on a novel EMT-driven fluorescent color switching breast cancer mouse model and intravital two-photon laser scanning microscopy. Specifically, the MMTV-PyMT, Rosa26-RFP-GFP, and Fsp1-Cre triple transgenic mouse model was used to monitor the conversion of RFP-positive epithelial cells to GFP-positive mesenchymal cells in mammary tumors under the control of the Fsp1 (ATL1) promoter, a gate-keeper of EMT initiation. RFP-positive cells were isolated from the tumors, sorted, and transplanted into mammary fat pads of SCID mice to monitor EMT during breast tumor formation. We found that the conversion from RFP- to GFP-positive and spindle-shaped cells was a gradual process, and that GFP-positive cells preferentially localized close to blood vessels, independent of tumor size. Furthermore, cells undergoing EMT expressed high levels of the HGF receptor, c-Met, and treatment of RFP-positive cells with the c-Met inhibitor, cabozantinib, suppressed the RFP-to-GFP conversion in vitro Moreover, administration of cabozantinib to mice with palpable RFP-positive tumors resulted in a silent EMT phenotype whereby GFP-positive cells exhibited reduced motility, leading to suppressed tumor growth. In conclusion, our imaging technique provides a novel opportunity for visualizing tumor EMT at the single-cell level and may help to reveal the intricacies underlying tumor dynamics and treatment responses. Cancer Res; 76(8); 2094-104. ©2016 AACR.

Characterization of a human tumorsphere glioma orthotopic model using magnetic resonance imaging.

Magnetic resonance imaging (MRI) is the imaging modality of choice by which to monitor patient gliomas and treatment effects, and has been applied to murine models of glioma. However, a major obstacle to the development of effective glioma therapeutics has been that widely used animal models of glioma have not accurately recapitulated the morphological heterogeneity and invasive nature of this very lethal human cancer. This deficiency is being alleviated somewhat as more representative models are being developed, but there is still a clear need for relevant yet practical models that are well-characterized in terms of their MRI features. Hence we sought to chronicle the MRI profile of a recently developed, comparatively straightforward human tumor stem cell (hTSC) derived glioma model in mice using conventional MRI methods. This model reproduces the salient features of gliomas in humans, including florid neoangiogenesis and aggressive invasion of normal brain. Accordingly, the variable, invasive morphology of hTSC gliomas visualized on MRI duplicated that seen in patients, and it differed considerably from the widely used U87 glioma model that does not invade normal brain. After several weeks of tumor growth the hTSC model exhibited an MRI contrast enhancing phenotype having variable intensity and an irregular shape, which mimicked the heterogeneous appearance observed with human glioma patients. The MRI findings reported here support the use of the hTSC glioma xenograft model combined with MRI, as a test platform for assessing candidate therapeutics for glioma, and for developing novel MR methods.

A lab-on-a-chip system integrating tissue sample preparation and multiplex RT-qPCR for gene expression analysis in point-of-care hepatotoxicity assessment.

A truly practical lab-on-a-chip (LOC) system for point-of-care testing (POCT) hepatotoxicity assessment necessitates the embodiment of full-automation, ease-of-use and sample-in-answer-out diagnostic capabilities. To date, the reported microfluidic devices for POCT hepatotoxicity assessment remain rudimentary as they largely embody only semi-quantitative or single sample/gene detection capabilities. In this paper, we describe, for the first time, an integrated LOC system that is somewhat close to a practical POCT hepatotoxicity assessment device - it embodies both tissue sample preparation and multiplex real-time RT-PCR. It features semi-automation, is relatively easy to use, and has sample-in-answer-out capabilities for multiplex gene expression analysis. Our tissue sample preparation module incorporating both a microhomogenizer and surface-treated paramagnetic microbeads yielded high purity mRNA extracts, considerably better than manual means of extraction. A primer preloading surface treatment procedure and the single-loading inlet on our multiplex real-time RT-PCR module simplify off-chip handling procedures for ease-of-use. To demonstrate the efficacy of our LOC system for POCT hepatotoxicity assessment, we perform a preclinical animal study with the administration of cyclophosphamide, followed by gene expression analysis of two critical protein biomarkers for liver function tests, aspartate transaminase (AST) and alanine transaminase (ALT). Our experimental results depict normalized fold changes of 1.62 and 1.31 for AST and ALT, respectively, illustrating up-regulations in their expression levels and hence validating their selection as critical genes of interest. In short, we illustrate the feasibility of multiplex gene expression analysis in an integrated LOC system as a viable POCT means for hepatotoxicity assessment.

Systems biology–based drug repositioning identifies digoxin as a potential therapy for groups 3 and 4 medulloblastoma

Systematic drug repositioning identifies digoxin as a potential treatment for groups 3 and 4 medulloblastoma. Digoxin on the brain Groups 3 and 4 medulloblastoma (MB) are highly heterogeneous in nature and have therefore proven difficult to target, resulting in corresponding meagre survival rates. Using a sophisticated systematic drug repositioning approach, Huang et al. identified the already-approved drug digoxin as a possible treatment for these MB subtypes. Application of digoxin to orthotopic patient-derived xenograft models produced an increase in survival; this increase in survival was further extended upon combining digoxin treatment with radiation, and, importantly, occurred at blood concentrations of digoxin that might be feasible in patients. These findings could mean a possible inroads in improving outcome for patients with these hard-to-treat cancers. Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Although outcomes have improved in recent decades, new treatments are still needed to improve survival and reduce treatment-related complications. The MB subtypes groups 3 and 4 represent a particular challenge due to their intragroup heterogeneity, which limits the options for “rational” targeted therapies. Here, we report a systems biology approach to drug repositioning that integrates a nonparametric, bootstrapping-based simulated annealing algorithm and a 3D drug functional network to characterize dysregulated driver signaling networks, thereby identifying potential drug candidates. From more than 1300 drug candidates studied, we identified five members of the cardiac glycoside family as potentially inhibiting the growth of groups 3 and 4 MB and subsequently confirmed this in vitro. Systemic in vivo treatment of orthotopic patient-derived xenograft (PDX) models of groups 3 and 4 MB with digoxin, a member of the cardiac glycoside family approved for the treatment of heart failure, prolonged animal survival at plasma concentrations known to be tolerated in humans. These results demonstrate the power of a systematic drug repositioning method in identifying a potential treatment for MB. Our strategy could potentially be used to accelerate the repositioning of treatments for other human cancers that lack clearly defined rational targets.

Targeting Brain-Adaptive Cancer Stem Cells Prohibits Brain Metastatic Colonization of Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) exhibits more traits possessed by cancer stem cells (CSC) than other breast cancer subtypes and is more likely to develop brain metastases. TNBC patients usually have shorter survival time after diagnosis of brain metastasis, suggesting an innate ability of TNBC tumor cells in adapting to the brain. In this study, we establish novel animal models to investigate early tumor adaptation in brain metastases by introducing both patient-derived and cell line-derived CSC-enriched brain metastasis tumorsphere cells into mice. We discovered astrocyte-involved tumor activation of protocadherin 7 (PCDH7)-PLCβ-Ca2+-CaMKII/S100A4 signaling as a mediator of brain metastatic tumor outgrowth. We further identified and evaluated the efficacy of a known drug, the selective PLC inhibitor edelfosine, in suppressing the PCDH7 signaling pathway to prohibit brain metastases in the animal models. The results of this study reveal a novel signaling pathway for brain metastases in TNBC and indicate a promising strategy of metastatic breast cancer prevention and treatment by targeting organ-adaptive cancer stem cells.Significance: These findings identify a compound to block adaptive signaling between cancer stem cells and brain astrocytes. Cancer Res; 78(8); 2052-64. ©2018 AACR.

Abstract 5460: Dual efficacy of Lazaroid U-74389G liposomes in glioblastoma mouse model

Purpose: Lazaroid U-74389G (LAZ) is a 21-aminosteroid that has radioprotective effect against radiation-induced lipid peroxidation. In-vitro antiproliferative effect was reported against Glioblastoma primary cell lines. The aim of this study was to use a liposomal formulation in a proof-of-concept efficacy study to evaluate LAZ radioprotective and antiproliferative effects in Glioblastoma mouse model. Methods: LAZ PEGylated liposomes (Lipo G) were developed in our laboratory. Lipo G size was 80-90 nm with Zeta potential of -22mv. Glioblastoma cell line U87-Lexpressing firefly luciferase reporter gene (100,000 cells in 2 μL) was injected intracranially in each male SCID hairless outbred mouse. The mice were randomized among 4 treatment groups (n=8-9, each): brain model (M) without treatment (control), radiation of 2Gy once a week (M+R), Lipo G at 5 mg/kg dose IP twice a week (M+L) and radiation with Lipo G (M+R+L). The treatment lasted three weeks. The tumor size was monitored using non-invasive bioluminescence imaging (BLI), in each mouse, expressed as the relative BLI photon intensity on the second and third week to that of the first measurement. The mice were sacrificed at the end of the third week and the whole brain was harvested for immunohistochemistry examinations. Lipid peroxidation of brain tissues was quantified by measuring malondialdehyde (MDA) as a surrogate biomarker. The survival was evaluated using Kaplan Meier analysis at P = 0.05. Results: The relative BLI intensity was 4002.03±1737.67, 2034±737.72, 1387.36±684.53 and 2498.89±2521.32 % for M, M+R, M+L and M+R+L, respectively. The tumor size of the M+L group was reduced by 65% compared to the control group. For the radiation treated groups (M+R and M+R+L), there was no significant difference in the tumor size suppression compared to the control group. MDA brain concentration in M+L and M+R+L groups was significantly less than that in M+R group (8.27±0.78 and 10.37±3.30 µM/gm vs. 23.09± 3.79 µM/gm). The survival mean was 22.67, 25.33, 25.22 and 27.13 days for M, M+R, M+R+L and M+L groups, respectively. The mean survival duration of LAZ treated groups (M+L and M+R+L) was significantly longer than that of the control group (27.13 and 25.22 day vs. 22.67 day, respectively). Conclusion: LAZ liposomal formulations administered at 5 mg/kg dose delivered a therapeutic concentration of LAZ to the brain for the efficacy of suppressing Glioblastoma tumor growth (65% reduction). The liposomal LAZ also protected the brain tissue from radiation-induced lipid peroxidation by reducing MDA concentration (lipid peroxidation surrogate) to 50%. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5460. doi:10.1158/1538-7445.AM2011-5460

Abstract 5161: Cell mechanics-cytoskeleton-membrane protein transduction loop mediates brain metastasis of breast cancer cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLnnBrain metastasis is the worst stage of breast cancer, as no therapy exists that prevents or eliminates breast cancer spreading to the brain. New therapeutic strategies depend on specific knowledge of tumor cell properties that allow breast cancer cells get through blood-brain barrier and grow in brain tissue. To provide information in this direction, we developed a microfluidic single-cell patch clamp microdevice to study the electrophysiological and biomechanical properties of human breast cancer brain metastatic cells for the first time. The results show that the brain metastatic cells possess higher membrane resistance and greater membrane stiffness compared to non-brain metastatic cells. The over-expression of HER2 and EGFR decrease the cell membrane resistance, while increase the cell stiffness, which correlates with the enhanced cell invasion through the in vitro blood-brain barrier and increased formation of brain metastatic loci in the xenograft mice. Gene expression analysis of the brain metastatic cells identifies cell membrane protein PCDH7 mediates the increased cell mechanics property functionally, and PCDH7 is also an enhancer of blood-brain barrier crossing and brain colonization for the brain metastatic cells, through the cytoskeleton rearrangement mechanism. This cell mechanics-cytoskeleton-PCDH7 protein loop mechanism may prove to be an attraction therapeutic target to block the brain metastasis of breast cancer.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5161. doi:10.1158/1538-7445.AM2011-5161

P3-17-02: Targeting the Autophagy Pathway for Drug Resistance of Breast Tumor-Initiating Cells.

Background: Our clinical data and experimental evidence reveal that the tumor-initiating cells (TICs) in the original tumor are intrinsically resistant to conventional chemotherapy and radiation therapy and greatly enriched in residual breast cancers after such treatments. We have published a gene expression signature of such breast TICs, and our pathway analysis on the gene signature suggests that the activation of autophagy pathway may be an intrinsic characteristic of the TICs. This motivates us to further investigate the role of the autophagy pathway in TICs self-maintenance and their resistance to hormonal and chemo therapies, as well as their response to TIC-targeted therapies. Methods: A collective 84 well-documented autophagy pathway genes were used to compare the activation of autophagy pathway in different microarray datasets, 1) flow-sorted CD44 + /CD24 −/low cancer cells vs. all other cells (representing 20 patients), and cancer-derived MSs vs. corresponding primary bulk tumors (representing 17 patients), 2) before vs. after letrozole and doxetaxel treatments (representing 30 patient pairs), 3) before vs. after letrozole treatment (representing 176 patient pairs), and 4) before vs. after lapatinib treatment (representing 115 patient pairs). We applied the Significance Analysis of Microarrays (SAM) algorithm to analyze the expression data of 211 normalized probes for the 84 genes. Low-density RTPCR array for the 84 genes was used to confirm the differential expressed genes on tumor tissues of 18 letrozol treated patient pairs. Two human-cancer-in-mouse triple-negative xenograft tumor lines were treated with Notch pathway inhibitor alone or in combining with docetaxel. The tumor growth, mammosphere formation efficiency, and the expression of autophagy marker proteins were evaluated. Results: Overall, the enrichment analysis of the 84-gene set in all the above microarray datasets showed that the autophagy genes are significantly enriched in the differential expressed genes. In particular, 28 out of the 84 autophagy genes are significantly up-regulated in the TICs populations while 5 other autophagy genes are down-regulated with the false discovery rate (FDR) less than 0.05. For the letrozole and doxetaxel treatment microarray analysis, 34 out of the 84 autophagy genes are significantly up-regulated in the after-treatment group (p Conclusion: Activation of the autophagy pathway in TICs is a promising target to combat the drug resistance of breast cancer to conventional systemic therapy. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-17-02.