bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 796: Reproductive hormones modulate cardioprotection from doxorubicin-induced cardiomyopathy in female spontaneously hypertensive rats
Authors: Pokrzywinski Kaytee , Rosen Elliot T. , Bonanno Julia L. , Aryal Baikuntha , Biel Thomas , Moshkelani Delaram , Mog Steven , Rao Ashutosh
Published: July 2017

Doxorubicin (Dox) causes cardiomyopathy, partially driven by the excess generation of mitochondrial reactive oxygen species (ROS). The incidence of Dox-induced cardiac stress is greater in prepubescent and postmenopausal females compared to reproductively mature women, suggesting that reproductive hormones may mediate cardioprotection. We investigated the role of exogenous hormone replacement therapy (HRT) and naturally cycling reproductive hormones in providing cardioprotection from Dox chemotherapy. Mammary adenocarcinoma SST-2 cells were implanted in spontaneously hypertensive rats (SHRs) that were either ovariectomized and received HRT, or left reproductively normal and maintained natural hormone cycling. HRT animals received time-release pellets containing 17-β-estradiol (E2), progesterone (P4), Tamoxifen (Tam) or combinations thereof, and were subsequently treated with Dox. Naturally cycling animals were treated with Dox, the iron chelator Dexrazoxane (Drz), or Dox+Drz. Tumor size was used to evaluate anticancer activity. Vaginal cytologies and circulating E2 and P4 levels were obtained. Serum cardiac troponin I (cTnI), histopathology and echocardiograms were used to assess cardiac health. Dox treated animals supplemented with E2 and P4 (alone) showed cardioprotection relative to the Dox treated animals. Dox+/-Drz exhibited sustained anti-tumor activity independent of endocrine status. After 13 days with Dox, all naturally cycling animals arrested in diestrus (lowest hormone levels). These results demonstrate that E2 and P4 have a role in providing cardioprotection from Dox therapy in SHRs, and that estrous cycle stage may provide a safety treatment window, potentially reducing the risk of cardiomyopathy in reproductively normal females. We are also currently analyzing RNA sequencing data obtained from heart tissues to achieve a better understanding of the molecular mechanisms of cardioprotection.

Citation Format: Kaytee Pokrzywinski, Elliot T. Rosen, Julia L. Bonanno, Baikuntha Aryal, Thomas Biel, Delaram Moshkelani, Steven Mog, Ashutosh Rao. Reproductive hormones modulate cardioprotection from doxorubicin-induced cardiomyopathy in female spontaneously hypertensive rats [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 796. doi:10.1158/1538-7445.AM2017-796
Abstract 809: A novel woodchuck model of hepatic artery infusion (HAI) with FACT complex targeted drug CBL0137
Authors: Kim Minhyung , Curtin Leslie I. , Powers Colin A. , Sexton Sandra , Gurova Katerina V. , Gudkov Andrei V. , Iyer Renuka V.
Published: July 2017

Background: The liver has unique anatomy in that most blood flow to normal hepatocytes is derived from the portal venous system, while liver tumors obtain their nutrient blood supply exclusively from the hepatic artery. Hepatic artery infusion (HAI) concentrates chemotherapy agents delivering them directly to the tumor bed with the first-pass effect. Although, HAI chemotherapy as a regional therapy has been used clinically for decades in the treatment of both primary and metastatic cancers of the liver, significant toxicity remains a concern, and clinical results have been mixed. The purpose of this study was to establish preclinical HAI model in a woodchuck and administer the FACT (facilitates chromatin transcription) complex targeted drug CBL0137.

Material and methods: Woodchuck hepatitis virus infected Eastern Woodchucks (Marmota monax) were used for the study. Hepatocellular carcinoma (HCC) developed spontaneously about 18 months after birth. The size of HCC was monitored with ultrasound every 2 weeks, and 4 woodchucks bearing over 2 cm tumors were used for the experimental group. Prophylactic cholecystectomy was performed first, and then a 2Fr. (ID=0.012”, OD=0.025”) vascular access port (VAP) was implanted in the gastroduodenal artery (GDA) after collateral vessel ligations. HAI was performed at room temperature via a VAP using 15mg/kg of CBL0137 at a volume of 3 mL/kg for 30 minutes, and tumor responses were investigated with MRI on day 0 (the surgery day of HAI port) and day 7 after HAI.

Results: Anatomy variations existed in the size and location of the supraduodenal artery or pancreatico-duodenal artery. 4/4 success rate of a port implantation was achieved without mortality, but 1/4 animals suffered post-operative bleeding at the port head site, attributed to coagulopathy secondary to hepatic dysfunction. And, 4/4 animals were partially anorexic for up to 7 days post-surgery. Decreased tumor volume and Ktrans (a measure of capillary permeability) were detected 7 days after single HAI with CBL0137.

Conclusion: Although the anticancer effect of CBL0137 on HCC was not clear because of a lack of the control group, we successfully implanted a VAP into the GDA of 4 woodchucks bearing unresectable HCC for HAI. The described woodchuck model may serve as a platform for identifying novel drugs and optimal conditions for the treatment of primary and metastatic liver tumors.

Citation Format: Minhyung Kim, Leslie I. Curtin, Colin A. Powers, Sandra Sexton, Katerina V. Gurova, Andrei V. Gudkov, Renuka V. Iyer. A novel woodchuck model of hepatic artery infusion (HAI) with FACT complex targeted drug CBL0137 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 809. doi:10.1158/1538-7445.AM2017-809
Abstract 810: Establishing patient derived preclinical in vitro and in vivo models of pediatric brain cancers
Authors: Yadavilli Sridevi , Kambhampati Madhuri , Gittens Jamila , Panditharatna Eshini , Stampar Mojca , Kilburn Lindsay B. , Magge Suresh , Packer Roger J. , Nazarian Javad
Published: July 2017

The recent surge in understanding genomic aberrations of some of the deadliest childhood brain cancers has highlighted the need for robust preclinical models. Such models will allow for robust drug screening and preclinical evaluation of efficacy, toxicity, and tumor penetrance in vivo. Given the rarity and importance of patient derived specimens, handling and processing methods are perhaps the most critical steps for successful establishment of viable and reproducible in vitro and in vivo models. Since specimen source varies (biopsy, autopsy, or cryo-preserved), processing methods should be refined to allow for optimal extraction of maximum numbers of viable cells from each specimen type. We have developed standardized procedures for handling and processing of tissue samples obtained from biopsy, autopsy, or cryo-preserved specimens as well as necropsy tissue obtained from existing xenograft models. Two processing methods for generating viable cell suspensions are described. The first method, which uses collagenase-DNAse mediated digestion of the tissue is efficient with bulky samples and can be used with tissues obtained at autopsy. The second method uses a commercially available enzymatic dissociation kit optimal for small volume samples such as biopsy, cryo-preserved and mouse necropsy specimens. We show that obtaining viable cell suspension from precious tumor tissue by these methods results in successful generation of pre-clinical in vitro and in vivo models of DIPG, pilocytic astrocytoma and medulloblastoma that represent the exact genetic makeup of the original patient tumor. We further demonstrate intracranial injections of these cells into P2 mice for generating orthotopic xenograft models of brainstem or cortical tumors. Our methods and results allow for rapid establishment of preclinical models using rare and valuable childhood brain tumor specimens. These pre-clinical models serve as valuable tools for understanding the molecular mechanisms of the disease, identifying targetable molecules, and screening of novel therapeutics.

Citation Format: Sridevi Yadavilli, Madhuri Kambhampati, Jamila Gittens, Eshini Panditharatna, Mojca Stampar, Lindsay B. Kilburn, Suresh Magge, Roger J. Packer, Javad Nazarian. Establishing patient derived preclinical in vitro and in vivo models of pediatric brain cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 810. doi:10.1158/1538-7445.AM2017-810
Abstract 811: Histological heterogeneity contributes to sunitinib resistance in clear cell renal cell carcinoma
Authors: Lichner Zsuzsanna , Saleeb Rola , Butz Henriett , Nofech-Mozes Roy , Riad Sara , Farag Mina , Kapus Andras , Yousef George
Published: July 2017

Introduction: The receptor tyrosine kinase (RTK) inhibitor sunitinib is the first line treatment for advanced clear cell renal cell carcinoma (ccRCC). Sunitinib inhibits angiogenesis via blocking signaling through VEGFR. About 80% of patients develop resistance after a drug-sensitive period. Molecular changes early in treatment may impact drug resistance, but are poorly understood.

Experimental Procedures: ACHN, 786-O and Renca cell lines were treated with 1 µM sunitinib. NSG mice were s.c. xenografted with the model cell lines and were treated with sunitinib at 40 mg/kg/day dose. mRNA expression was screened using Illumina HT-12 bead chip array and miRNA expression was assessed by Nanostring nCounter assay. R statistical packages were used for data processing. Reactome and miRPath softwares were used for downstream analysis.

Results: Sunitinib treatment of ccRCC xenografts led to several early changes in tumor histology, such as the emergence of live tumor areas within the necrotic spaces. These areas showed membranous staining for E-cadherin, and β-catenin, while the rest of the tumor and vehicle-treated tumors were negative. In vitro model cell lines developed cancer spheroids when treated with sunitinib. Cancer spheroids were highly tumorigenic and metastatic, and expressed several established cancer stem cell markers. ccRCC cancer spheres, but not the 2D adherent cells, showed membranous staining for E-cadherin and β-catenin; similarly to the live tumor areas observed in

in vivo sunitinib treatment. In vitro inhibition of E-cadherin by EGTA or by siRNA, interfered with viability of sunitinib treated ccRCC cell lines.

Conclusions: Sunitinib treatment causes early phenotypic changes of the tumor in vivo and in vitro. The formation of highly metastatic and tumorigenic cancer spheres in model cell lines is the most prominent effect in vitro. We provide preliminary evidence that sunitinib induced in vitro cancer spheres and the live tumor areas that survive within necrotic patches of the sunitinib-treated xenografts, are related. Finally, membranous expression of E-cadherin enhances the survival of ccRCC cell lines under sunitinib treatment.

Citation Format: Zsuzsanna Lichner, Rola Saleeb, Henriett Butz, Roy Nofech-Mozes, Sara Riad, Mina Farag, Andras Kapus, George Yousef. Histological heterogeneity contributes to sunitinib resistance in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 811. doi:10.1158/1538-7445.AM2017-811
Abstract 833: Alginate hydrogels for three-dimensional culture and real-time monitoring of cancer cell metabolism and radiation response
Authors: Kesarwala Aparna H. , Carter Jenna L. , Read Graham H. , Miura Natsuko , Yamamoto Kazutoshi , Mitchell James B. , Krishna Murali C.
Published: July 2017

The purpose of this study was to characterize a three-dimensional calcium alginate culture system for real-time monitoring of cancer cell metabolism and radiation response. HCT116 colorectal cancer cells were encapsulated in 2.5% calcium alginate using a custom electrostatic rig adapted from a previously published system. Bead diameter was a function of voltage and alginate concentration: 4 x 107 cells/mL encapsulated at 4 and 8 kV resulted in mean diameters of 880 and 584 µm, respectively. The distribution of bead radii for all voltages showed positive excess kurtosis (4 kV = 1.6, 8 kV = 3.0) and 72.9% of 4 kV and 77.2% of 8 kV bead diameters fell within one standard deviation of the mean. Encapsulated cell viability was assessed by staining with Trypan Blue and clonogenic survival assays (CSAs). The baseline viability of cells immediately after encapsulation was 93.2%, which decreased 2 days after a single 10 Gy dose (90.7%, p<0.05). After two weeks of continuous culturing, 84.1% of non-irradiated and 73.8% of irradiated encapsulated cells remained viable (p<0.003). Pimonidazole staining demonstrated the presence of hypoxic cores proportional to bead size. CSAs of 8 kV beads showed a dose modifying factor (DMF) of 1.06 relative to cells grown in a monolayer, while larger 4 kV beads showed a DMF of 1.58. Mean oxygen consumption rate rose with increasing number of 8 kV beads/well: 1 bead, 35.5; 2 beads, 97.4; 3 beads, 175.5; 4 beads, 209.9; 2.0 x 104 monolayer cells, 92.6 (all pmol/min, ~8.2 x103 cells/bead, p<0.005). Hyperpolarized 13C-NMR spectroscopy of 400 uL of 8 kV beads containing 1.2 x 108 cells/mL showed a detectable conversion of [1-13C]-pyruvate to [1-13C]-lactate; 1 hour after a single 10 Gy dose, the lactate:pyruvate ratio decreased by 25%. Unpaired 2-tailed Student’s t-tests were used to determine significance between and one-way ANOVA was used to determine significance among groups with α set at p≤0.05. These data demonstrate the versatility of alginate hydrogels for real-time metabolic and radiation response studies which are non-invasive, higher throughput, and lower cost compared to in vivo systems. Future directions include additional metabolic flux analysis and hyperpolarized 13C-NMR spectroscopy to further investigate the cancer cell metabolic response to drug and/or radiation therapy.

Citation Format: Aparna H. Kesarwala, Jenna L. Carter, Graham H. Read, Natsuko Miura, Kazutoshi Yamamoto, James B. Mitchell, Murali C. Krishna. Alginate hydrogels for three-dimensional culture and real-time monitoring of cancer cell metabolism and radiation response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 833. doi:10.1158/1538-7445.AM2017-833
Abstract 859: Epigenetic regulation of epithelial-mesenchymal transition in esophageal squamous cell carcinoma
Authors: Chen Chen , Zhao Ming , Huang Xiaojie , Wang Xiang , Liu Wenliang , Chen Mingjiu , Yin Bangliang , Li Zhi , Yuan Yunchang , Lu Qianjin , Yu Fenglei
Published: July 2017

Epithelial-mesenchymal transition (EMT) is a developmental program, which is associated with tumor progression and metastasis. Here we investigated the DNA methylation and histone modifications in the regulation of EMT in esophageal squamous cell carcinoma (ESCC). The RNA-Seq analysis identified a total of 6150 differentially expressed genes (3423 up-regulated and 2727 down-regulated). The GO terms showed that these genes belonged to several molecular functions and biological pathways. The abnormal expression of key EMT genes, some of Sox family genes and EZH2 significantly related to patient survival. The MeDIP-Seq analysis identified differentially methylated regions (DMRs) on the whole genome level with focal hypermethylation and widespread global DNA hypomethylation. The gene ontology analysis showed that the DMRs related genes belonged to several different ontological domains, such as EMT progression, cell cycle, adhesion, proliferation, and apoptosis. The results of the bisulfite-sequencing confirmed the EMT gene DMRs identified by MeDIP-Seq. However, further analyses showed that the EMT gene DMRs only related to gene expression, but not the patient survival. ChIP-Seq was then performed to observe the relationship between histone methylation and gene expression. A total of 295 genes were found to correlate with H3K27 tri-methylation (H3K27me3) , and a certain number of key EMT genes, such as ZEB1/2,AREG, GATA4,and CDH8 were identified. Further study showed that Ezh2-mediated H3K27me3 regulate the expression of key EMT genes. Ablation of Ezh2 expression prevents EMT, whereas forced expression of Ezh2 restores EMT. Sox4 gene could directly regulate Ezh2 expression, tri-methylated H3K27me3 for EMT gene repression. Taken together, our results suggest that both of the DNA methylation and H3K27me3 modification involved in the regulation of EMT in ESCC. Sox4-Ezh2-mediated H3K27me3 marks associate with key EMT gene regulation, representing an important epigenetic EMT signature.

Note: This abstract was not presented at the meeting.

Citation Format: Chen Chen, Ming Zhao, Xiaojie Huang, Xiang Wang, Wenliang Liu, Mingjiu Chen, Bangliang Yin, Zhi Li, Yunchang Yuan, Qianjin Lu, Fenglei Yu. Epigenetic regulation of epithelial-mesenchymal transition in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 859. doi:10.1158/1538-7445.AM2017-859
Abstract 861: Olfactomedin 4 downregulation is associated with enhancing epithelial-mesenchymal transition in human benign prostate cells and prostate cancer cells
Authors: Li Hongzhen , Liu Wenli , Zhu Jianqiong , Chin Kay , Rodriguez-Canales Jaime , Rodgers Griffin P.
Published: July 2017

We have previously reported that lost or reduced olfactomedin 4 (OLFM4) expression is caused by deletions within the OLFM4 gene in 10-25% of all human prostate-cancer samples, and that its expression is lost in more than 58% of advanced prostate-cancer samples. Therefore, we sought to study the role of epigenetic factors in the regulation of OLFM4 gene expression in normal prostate, as well as prostate-cancer, cell lines. Pyrosequencing of genomic DNA extracted from normal prostate or prostate-cancer tissue cells demonstrated that three of the eight CpG sites in the OLFM4 gene promoter were significantly hypermethylated in prostate cancer compared with normal tissue. Exposure to the methylation inhibitor 5-azadeoxycytidine (5-Aza 5 μM) significantly increased OLFM4 expression in RWPE1 benign prostate cells. Knockdown of OLFM4 expression in RWPE benign prostate cells reduced autophagy and enhanced the epithelial-mesenchymal transition (EMT). Restoration of OLFM4 expression in metastatic prostate-cancer cells lacking OLFM4 inhibited cell growth in both in vitro culture models and an in vivo xenograft tumor model, and also inhibited EMT marker gene expression in these cells. 5-Aza induced OLFM4 expression in PC-3 prostate cancer cells and OLFM4 shRNA transfected PC-3 cells affects 5-Aza mediated cell proliferation compared to control-shRNA transfected PC-3 cells. Taken together, our results suggest that OLFM4 inhibits the EMT program in both normal prostate and prostate-cancer cells, and may play a critical role in regulating the EMT program during the progression of cancer. They also suggest that modulation of OLFM4 expression might be a novel molecular target for advancing prostate-cancer therapies.

Citation Format: Hongzhen Li, Wenli Liu, Jianqiong Zhu, Kay Chin, Jaime Rodriguez-Canales, Griffin P. Rodgers. Olfactomedin 4 downregulation is associated with enhancing epithelial-mesenchymal transition in human benign prostate cells and prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 861. doi:10.1158/1538-7445.AM2017-861
Abstract 863: Neuronal splicing regulatory factors are upregulated in models of epithelial-mesenchymal transition
Authors: Khodor Yevgenia L. , Froese Julia , Gertler Frank , Weinberg Robert A. , Burge Christopher B.
Published: July 2017

Epithelial-mesenchymal transition (EMT) is an essential developmental program through which epithelial cells lose their polarity and junctions with neighboring cells and acquire migratory and invasive properties. Cancer cells hijack at least a portion of the EMT program to increase their invasive properties, acquire resistance to senescence, and metastasize. Major changes in cell signaling, transcriptional and post-transcriptional regulation occur during EMT. To study post-transcriptional changes we characterized gene expression and splicing across EMT in 3 cell culture models of EMT: the luminal epithelial MCF7ras breast cancer cell line with EMT induced by SLUG+SOX9 and in basal human mammary epithelial (HMLEras) cells, with EMT induced by either SNAIL or ZEB1. RNA-seq was performed at 2-day intervals across these transitions. We observed broad overlaps between these systems, with 821 genes that changed significantly in all three. In all three time courses, genes associated with cell cycle and growth decreased in expression, while genes associated with signaling, cell motion, adhesion, extracellular matrix organization, and differentiation increased. Intriguingly, genes associated with neurogenesis and angiogenesis were also induced in the MCF7 and HMLER-Snail time courses. Those include known axon guidance and angiogenic factors in the Robo and Slit families. We also found many alternative splicing events shared in all 3 systems, including 272 skipped exons. Sequence analysis of regions around these exons revealed binding motifs for canonical splicing regulators, including ESRP1 and ESRP2 and the RBFOX protein family, which are known to play a role in EMT. While most genes encoding RNA binding proteins decreased in expression in EMT, a small subset consistently increased in expression, including some that matched our motif analysis. Specifically, several splicing factors associated with nervous system development were upregulated, including factors of the QKI and NOVA families. We have validated the increased expression of QKI and NOVA1 proteins during EMT in vitro, and of QKI and NOVA2 in tumor sections. Further, we found that perturbation of QKI expression affects the motility of mesenchymal cancer cells in culture. Together, our results point to the existence of a program of alternative splicing shared between neuronal and mesenchymal cells.

Citation Format: Yevgenia L. Khodor, Julia Froese, Frank Gertler, Robert A. Weinberg, Christopher B. Burge. Neuronal splicing regulatory factors are upregulated in models of epithelial-mesenchymal transition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 863. doi:10.1158/1538-7445.AM2017-863
Abstract 864: Alternated activation of TGFβ pathway and differential expression of epithelial to mesenchymal transition transcription factors in relation to lymph node metastasis in two subtypes of breast cancer tumors
Authors: Ortega-Hernandez Victoria , Gajardo-Meneses Patricia , Fernandez Wanda , Carvallo Pilar
Published: July 2017

Breast cancer is the leading cause of cancer death among women worldwide, being distant metastases the main cause of death. Epithelial to mesenchymal transition (EMT) has been implicated in promoting cancer invasion and metastasis. This process can be induced by TGFβ through the expression of transcription factors: TWIST, SNAIL, SLUG and ZEB1, which expression may be promoted by different signaling pathways such as SMAD2/SMAD3, PI3K/AKT and ERK/MAPK. The expression of EMT transcription factors has been previously studied in several cancer cell lines, although no studies have been performed in breast cancer tumors. In this study, we analyzed by immunohistochemistry the expression of the four transcription factors, and the signal transduction key proteins p-SMAD2 (ser465/467), p-SMAD2L (ser245/250/,255) and p-AKT (ser473), in 107 breast cancer tumors. We found expression of at least one transcription factor in all triple negative tumors (n=24), while only 70% luminal tumors showed expression of at least one of these proteins. Expression of ZEB1, SNAIL and SLUG was observed in tumors with metastases to lymph node, whereas the majority of tumors expressing TWIST were negative for lymph node status. All triple negative tumors with an active state of the signal transduction pathway TGFβ/SMAD showed expression of at least one transcription factor, whereas 65% of luminal tumors with an active state of the same pathway showed no expression of these transcription factors. Active state of PI3K/AKT pathway was observed only for luminal tumors as it has been described. Sixty-three percent of tumors having this pathway active showed expression of at least one transcription factor. In conclusion we found differential expression of EMT transcription factors in relation to lymph node status and a differential active state of signaling pathways in relation to tumoral subtype of breast cancer tumors. Our results suggest a different mechanism of EMT induction by TGFβ in relation to two tumor’s subtypes and lymph node metastasis.

Citation Format: Victoria Ortega-Hernandez, Patricia Gajardo-Meneses, Wanda Fernandez, Pilar Carvallo. Alternated activation of TGFβ pathway and differential expression of epithelial to mesenchymal transition transcription factors in relation to lymph node metastasis in two subtypes of breast cancer tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 864. doi:10.1158/1538-7445.AM2017-864
Abstract 868: GRK5 promotes a mesenchymal transition in prostate cancer
Authors: Black Joseph B. , Mahmud Iqbal , Purayil Hamsa Thayele , Daaka Yehia
Published: July 2017

Locally advanced and metastatic prostate cancer is incurable, despite decades of research targeting the activity of the androgen receptor (AR). Our lab previously identified G protein-coupled receptor 5 (GRK5) for its ability to regulate PCa progression, independent of AR activity. GRK5 partially partitions to the nucleus, wherein it has been shown to regulate the transcriptome in non-PCa models. To globally elucidate the mechanistic impact of GRK5 on PCa progression, we are investigating the impact of GRK5 on the PCa transcriptome. We hypothesize that GRK5’s regulation of the PCa transcriptome promotes tumor progression. To assay the effect of GRK5 on the PCa transcriptome, RNA sequencing was performed in two cell lines: PC3 Control (PC3 shGFP) and PC3 GRK5 Knockdown (PC3 shGRK5). Ontologic analysis identified the epithelial–mesenchymal transition (EMT) as being affected when GRK5 is depleted. The epithelial–mesenchymal transition (EMT) is highly associated with promoting PCa progression and chemoresistance, thus we selected this pathway for further investigation. Confirmatory western blot analysis and quantitative PCR (qPCR) validated that depleting GRK5 suppresses the expression of the mesenchymal markers Vimentin and N-Cadherin. Similarly, overexpression of GRK5 increases the expression of these mesenchymal markers. Stable overexpression of GRK5 promotes cells to develop a spindle-like morphology, indicative of a mesenchymal state. GRK5 mediates this mesenchymal transition through increasing the expression of the EMT transcription factor, Twist1. Further analysis identifies that GRK5 activity promotes in vitro invasion. Cell lines overexpressing GRK5 demonstrate an increase in resistance to docetaxel, the mainstay chemotherapy for advanced PCa. Overexpression of mutated forms of GRK5 that are relegated to the nucleus are able recapitulated all aforesaid changes, arguing that the nuclear activity of GRK5 mediates this effect. Collectively, this data presents a novel mechanism promoting PCa progression, independent of AR activity. Future work seeks to assay the ability of GRK5 to promote metastasis in vivo in mouse models.

Note: This abstract was not presented at the meeting.

Citation Format: Joseph B. Black, Iqbal Mahmud, Hamsa Thayele Purayil, Yehia Daaka. GRK5 promotes a mesenchymal transition in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 868. doi:10.1158/1538-7445.AM2017-868
Abstract 87: Treatment schedules influence acquisition of Doxorubicin resistance through epigenetic mechanism in breast cancer cells
Authors: Ponnusamy Logeswari , Mahalingaiah Prathap Kumar S. , Singh Kamaleshwar P.
Published: July 2017

Breast cancer is the leading cause of cancer related mortality in women. Chemotherapy resistance is a major limitation in clinical treatment of breast cancer. Mounting evidence implicates the epigenetic aberrations in acquired chemoresistance. However, the influence of treatment schedules and associated epigenetic changes on acquisition of doxorubicin resistance in breast cancer is not clear. Therefore, objective of this study was to evaluate the impact of treatment schedules on acquisition of doxorubicin resistance and the molecular mechanistic basis of this process in breast cancer cells. To address this question, two different treatment strategies viz, continuous exposure and intermittent exposure were used to generate doxorubicin resistant cells lines from ER-positive MCF-7 and triple negative MDA-MB-231 breast cancer cell lines. The level of resistance against doxorubicin and the temporal sequence of molecular changes associated with the resistance phenotype were evaluated at 4 months and 18 months’ following exposure to doxorubicin. Results revealed that intermittent exposure to doxorubicin resulted in significantly higher level of resistance as compared to continuous exposure. ER-positive MCF-7 cells developed relatively earlier and high level of resistance when compared to MDA-MB-231 cells. Additionally, the resistance to doxorubicin-induced cytotoxicity was associated with transient acquisition of EMT and CSC-like phenotype in breast cancer cells around 4 months following doxorubicin. Gene and protein expression analysis further revealed temporal changes in the expression of epigenetic regulatory genes and histone modifications respectively. The temporal changes in expression of genes were positively correlated with doxorubicin exposure time during resistance development. In summary, the result of this study for the first time suggests that the treatment schedules influence the acquisition of doxorubicin resistance potentially through epigenetic mechanisms. The findings of this study will be helpful in chemotherapy of breast cancer to achieve the best clinical result

Note: This abstract was not presented at the meeting.

Citation Format: Logeswari Ponnusamy, Prathap Kumar S. Mahalingaiah, Kamaleshwar P. Singh. Treatment schedules influence acquisition of Doxorubicin resistance through epigenetic mechanism in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 87. doi:10.1158/1538-7445.AM2017-87
Abstract 888: Volumetric reconstruction of targeted nanoparticles for superparamagnetic relaxometry
Authors: Thrower Sara L. , Mathieu Kelsey , Stefan Wolfgang , Aburto R. Romero , Lu Zhen , Bast Robert C. , Sovizi Javad , Fuentes David , Hazle John D.
Published: July 2017

Superparamagnetic relaxometry (SPMR) is an emerging technology that uses the unique magnetic properties of superparamagnetic iron oxide nanoparticles (SPIONs) to detect cancer cells. In order to estimate tumor locations from raw MRX data, we developed an L1 reconstruction algorithm under the assumption that early stage disease is sparsely distributed throughout the anatomy. The approach was previously validated in phantom datasets of known signal locations. Advantages of our method are that the solver does not require the user to input prior information regarding the expected number of tumors or their approximate locations. Additionally, the solver reconstructs a volumetric distribution of detected sources within the field of view. To validate the algorithm for use in preclinical settings, SPMR was performed on SKOV3 ovarian tumor bearing mice (n = 3) with the MRX device over time following an intratumoral injection of anti-Her2 antibody-conjugated 25nm SPIONs (Senior Scientific LLC). The SPMR data was reconstructed with our sparse solver and was found to be highly correlated (r = 0.9978) with the results generated by the commercial software that accompanies the MRX instrument (MSA). Additionally, segmentation of the reconstruction revealed a strong signal (2.0·106 pJ/T) in the area of the tumor and almost no signal in areas outside of the tumor (0.077 pJ/T) at four hours after injection. This result was consistent with our prior observations which have revealed that a large fraction of intratumorally-injected nanoparticles remain localized within the tumor for several hours after injection. Furthermore, these results were consistent with SPMR data collected by measuring tissue samples excised 24 hours after injection, of which the tumor had the highest signal. Thus, our sparse reconstruction algorithm was able to return the expected results without prior information regarding the location of nanoparticles. Future work will focus on quantifying the uncertainty in our reconstruction method, as well as characterizing its stability with increasingly complex nanoparticle distributions and detectability limits. In conclusion, this work represents an important advancement of the SPMR technology by allowing for volumetric reconstructions of bound nanoparticles from in vivo data.

Citation Format: Sara L. Thrower, Kelsey Mathieu, Wolfgang Stefan, R. Romero Aburto, Zhen Lu, Robert C. Bast, Javad Sovizi, David Fuentes, John D. Hazle. Volumetric reconstruction of targeted nanoparticles for superparamagnetic relaxometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 888. doi:10.1158/1538-7445.AM2017-888
Abstract 900: Tumor Treating Fields (TTFields) affect human glioma cell migration, invasion and adherence properties in vitro
Authors: Garcia-Carracedo Dario , Schneiderman Rosa S. , Zeevi Einav , Shteingauz2 Anna , Giladi Moshe , Voloshin Tali , Porat Yaara , Munster Mijal , Blat Roni , Kirson Eilon D. , Weinberg Uri , Palti Yoram
Published: July 2017

TTFields, an approved treatment modality for glioblastoma, are delivered via continuous, noninvasive application of low intensity, intermediate frequency alternating electric fields. The antimitotic effects of TTFields have been extensively investigated. We investigated other processes that may be affected by TTFields such as human glioma cell migration and cellular invasion properties. Four human glioma cell lines were treated with TTFields using the inovitro system. Cell migration rates were measured using wound healing assays. Invasion assays were performed using a modified Matrigel coated Boyden chamber. Cell adhesion assays were performed during TTFields treatment and compared to untreated controls. At the end of TTFields’ treatment, adhered cells were trypsinized and counted. A cells de-adhesion assay was performed following 24 and 72 hours of TTFields application with the outcome being the number of cells removed after varying times of trypsinization. Application of TTFields in-vitro led to a significant reduction in cell migration velocity compared with untreated control cells. Furthermore, comparison of the anti-migratory efficacy of TTFields applied from 2 directions vs. a single direction revealed that the single direction approach applied perpendicularly to the course of migration was more effective than TTFields applied in 2 directions or in a single parallel direction. Glioma cells invasion was significantly reduced compared to untreated cells in all tested cell lines. Cell adhesion to the substrate (fibronectin) was significantly reduced when exposed to TTFields. On the other hand, cell de-adherence following TTFields treatment took significantly longer time of trypsinization. Our results suggest that human glioma cell motility is impaired by exposure to TTFields. Both alterations in cells adherence and de-adherence during exposure to TTFields may contribute to reduction in cell motility. Further studies are needed to elucidate the mechanism by which TTFields disrupts cellular motility in glioma cancer cells.

Citation Format: Dario Garcia-Carracedo, Rosa S. Schneiderman, Einav Zeevi, Anna Shteingauz2, Moshe Giladi, Tali Voloshin, Yaara Porat, Mijal Munster, Roni Blat, Eilon D. Kirson, Uri Weinberg, Yoram Palti. Tumor Treating Fields (TTFields) affect human glioma cell migration, invasion and adherence properties in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 900. doi:10.1158/1538-7445.AM2017-900
Abstract 919: Identification of stem cells in stomach corpus and antrum
Authors: Matsuo Junichi , Yamamura Akihiro , Yeoh Khay Guan , Osato Motomi , Ito Yoshiaki
Published: July 2017

Gastric cancer is still relatively poorly understood, because of the lack of knowledge on stem cells in stomach. By radio-labeling experiments and electron microscopy, rapidly proliferating stem cells have been suggested in isthmus of both corpus (which is just below pit cell zone) and antrum (which is a quarter above the base) (1). In addition, there have been reports indicating the proliferation capacity under certain conditions in a subset of chief cells present at the base in corpus (2). We reported earlier that 270 bp Runx1 enhancer element (termed aR1) that drives the expression of Runx1 in hematopoietic stem cells (3). We found that eR1 also marks the cells in these three locations of stomach (4). In antrum, the stem cell marker, Lgr5, has been reported to mark the cells at the base of antrum but not in corpus (5). eR1+ and Lgr5+ stem cell in antrum appears to be different. It will be interesting to find out the difference between these two types of stem cells. Transgenic mouse was generated that harbors eR1 linked to heterologous promoter and Cre recombinase (eR1-CreERT2). By treating the mouse with tamoxifen, we showed lineage tracing from eR1+ cells in the isthmus of both corpus and antrum that replenish entire glands. We expressed K-rasG12D in eR1+ cells in isthmus and observed antralization of corpus epithelium that is similar to precancerous lesion in human stomach after H pylori infection. On the other hand, eR1+ chief cells rarely multiply. However, they robustly proliferate after tissue damage or oncogene expression. When K-rasG12D is activated in eR1+ chief cells, they show retrodifferentiation to show SPEM (spasmolytic polypeptide expressing metaplasia) phenotype. eR1+ chief cells appear to be reserve stem cells that can replace cells after tissue damage. We now have capability to activate or inactivate genes of interest in the stomach stem cells. We are examining stepwise carcinogenesis in stomach. From eR1+ cells in isthmus of both corpus and antrum, we are generating organoids. We will describe interesting difference in the properties of organoids generated from eR1+ cells from corpus and those from antrum.  (1) Karam and Leblond., The Anatomical Record 1993; 236: 259-273. (2) Goldenring et al., Gastroenterology 2011; 138: 2207-2210. (3) Ng et al., Stem Cells 2010; 28: 1869-81. (4) Matsuo et al., Gastroenterology 2016; PMID: 27670082. (5) Barker et al., Cell Stem Cell 2010; 6: 25-36.

Citation Format: Junichi Matsuo, Akihiro Yamamura, Khay Guan Yeoh, Motomi Osato, Yoshiaki Ito. Identification of stem cells in stomach corpus and antrum [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 919. doi:10.1158/1538-7445.AM2017-919
Abstract 92: Acquisition of temozolomide resistance: Identification of a new drug tolerant stage in glioblastoma cells
Authors: Rabé Marion , Janati Hicham , Dumont Solenne , Thibault-Carpentier Christelle , Clairambault Jean , Vallette François M. , Gratas Catherine
Published: July 2017

Glioblastoma multiforme (GBM) are the most aggressive and common brain tumors in adults. Despite surgery and combined radio-chemotherapy with temozolomide (TMZ), tumor reccurence always occurs. The median survival time for patients diagnosed with GBM is about 14 months with less than 5% survival at 5 years. Today the main marker of TMZ resistance is the methylation status of MGMT promoter. Patients with a methylated promoter usually have a better response to treatment than patients with an unmethylated promoter. Indeed MGMT is an enzyme involved in DNA repair mechanisms that abrogates TMZ effects. However, in clinical trials targeting the MGMT enzyme, median survival of patients was not improved. It is thus essential to decipher the mechanisms involved in the acquisition of TMZ resistance to identify new therapeutic targets. To achieve this goal TMZ resistant cells were generated by continuous treatment of the U251 human glioblastoma cell line. These cells are sensitive to TMZ and do not express MGMT. We performed transcriptomic analysis by RNA-Seq on U251 treated with TMZ (50µM) for different time and selected differentially expressed genes. We also evaluated target genes expression in single cells by RT-qPCR using C1-HD-Biomark technology (Fluidigm). Transcriptome profiling allowed to identify a transient phase with TMZ tolerant cells before acquisition of complete resistance. These cells are characterized by a modified morphology and a no proliferative state. In this population we identified a subset of genes with the same transient overexpression. Similar results were observed in other glioblastoma cell lines and under other stress conditions. In contrast, stable expression of MGMT appeared later with the emergence of the TMZ resistant population. Interestingly single cell qPCR showed that MGMT expression in the resistant cells could not be explained by clonal selection of MGMT positive cells. Drug screening on the TMZ tolerant cells revealed a potent killing activity of histone deacetylating agents (HDAC inhibitors). In conclusion, we have shown that glioblastoma cells become resistant after a transient state of TMZ tolerance. Identification of this singular population highlights new molecular targets and a new therapeutic window. Targeting these tolerant cells could avoid emergence of resistance and tumor recurrence, thereby patients survival could be improved.

Citation Format: Marion Rabé, Hicham Janati, Solenne Dumont, Christelle Thibault-Carpentier, Jean Clairambault, François M. Vallette, Catherine Gratas. Acquisition of temozolomide resistance: Identification of a new drug tolerant stage in glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 92. doi:10.1158/1538-7445.AM2017-92
Abstract 920: ICAM-1(CD54) is involved in the osteogenic differentiation of mesenchymal stem cells and affects the progression of osteosarcoma cells
Authors: Zhang Yidan , Piperdi Sajida , Zaphiros Nikolas , Koirala Pratistha , Ren Tingting , Roth Michael , Gill Jonathan , Hoang Bang , Geller David , Yang Rui , Zhang Wendong , Du Xiuquan , Zhang Jinghang , Gorlick Richard
Published: July 2017

Purpose: The aim of this study is to validate the relationship between the expression of ICAM-1(CD54), a cell surface glycoprotein, and osteogenic differentiation of mesenchymal stem cells (MSCs) and the role of ICAM-1 in the progression of osteosarcoma (OS) cells.

Experimental Design: To compare the gene expression profiles between MSCs and MSCs after osteogenic induction, a microarray analysis was first performed. The differences in expression of specific genes including ICAM-1 were confirmed using western blot and flow cytometry. MSCs were sorted into two groups of cells by CD105+CD44+CD54+ and CD105+C44+CD54- and properties of these cells were compared. Expression of ICAM-1 among tumor cell lines and xenograft OS cells were tested at the protein level. ICAM-1 was stably, constitutively overexpressed in multiple cell types. Differences in osteogenic differentiation potential between normal cell lines and transformed cell lines were studied using alizarin red S staining. Motility, proliferation and invasion as well as the metastatic potential of the transformed and normal cells were evaluated.

Results: The initial microarray analysis revealed that ICAM-1 expression was significantly increased in MSCs after osteogenic induction for 15 days (p=0.042). Consistent results were achieved in western blot and flow cytometry analysis. Further microarray analysis between CD105+CD44+CD54+ and CD105+C44+CD54- groups determined that NF-kB signaling pathway was differentially activated. As for OS cells, ICAM-1 is not expressed at high level among OS tumor cell lines and is inconsistently expressed in xenograft OS cells. Enhanced osteogenic differentiation potential was observed in ICAM-1 overexpressing cells. Although there were no major difference in in vitro motility, proliferation or invasion tests, substantially less lung nodules occurred with transformation as compared to the normal control.

Conclusions:

ICAM-1 is involved in the osteogenic differentiation of mesenchymal stem cells and its down regulation may be involved in the progression of osteosarcoma cells.

Citation Format: Yidan Zhang, Sajida Piperdi, Nikolas Zaphiros, Pratistha Koirala, Tingting Ren, Michael Roth, Jonathan Gill, Bang Hoang, David Geller, Rui Yang, Wendong Zhang, Xiuquan Du, Jinghang Zhang, Richard Gorlick. ICAM-1(CD54) is involved in the osteogenic differentiation of mesenchymal stem cells and affects the progression of osteosarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 920. doi:10.1158/1538-7445.AM2017-920