bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5792: Development of cell line derived organoids to evaluate stroma targeted therapies in pancreatic cancer
Authors: Hall Bradley , Santhamma Bindu , Cannon Andrew , Bhatia Rakesh , Kumar Sushil , Are Chandrakanth , Nair Hareesh , Nickisch Klaus , Batra Surinder
Published: July 2017

Background: Most solid tumors have extensive stroma that not only facilitates the tumor progression but also impedes the delivery of the chemotherapeutic agents. Due to lack of any in-vitro system, presently it is difficult to evaluate any stroma-targeted therapies. Therefore, we developed an organoid system using labeled pancreatic cancer and stellate cell lines.

Methods: Murine (FC 1295 and imPSCc-2) cell lines cultured in different combinations were grown as an organoid system using matrigel. The organoids, starting day four were treated with either gemcitabine or EC359, a novel mifepristone derived steroidal cytotoxic agent that targets tumor stroma, or both in combination. qRT-PCR analysis of activated stroma signature genes was performed on the mRNA isolated from different treatment groups. H&E, immunohistochemistry, and western blot analysis were performed to further validate our findings.

Results: Histologically, these cell line derived organoids develop ductal structures surrounded by fibroblast as seen in the pancreatic tumors. Immunohistochemistry demonstrated alpha-SMA staining of imPSCc-2 while FC 1295 cells were stained positive for CK19 and Na/K-ATPase. Gemcitabine treatment of the organoids resulted in 98% relative reduction in GFP fluorescence while EC359 decreased the fluorescence by 46% as compared to control. qRT-PCR demonstrated significant reduction (p<0.01) in the expression of activated stroma associated genes such as COL1A1, POSTN, SPARC, COL3A1, COL5A2 and THBS2. However, there was a significant increase in the expression of COL10A1, suggesting the differential regulation of stroma associated genes to stroma-targeted therapies. Further, western blot analysis demonstrated that EC359 reduced the expression of markers of activated stroma including alpha-SMA, and vimentin.

Conclusions: Our novel cell line derived organoid model can be utilized for fast, and inexpensive evaluation of stroma-targeted therapies. Also, our study demonstrates that gemcitabine and a novel mifepristone-derivative can be used as combination therapy to simultaneously target cancer cells and associated stroma for the treatment of pancreatic cancer.

Citation Format: Bradley Hall, Bindu Santhamma, Andrew Cannon, Rakesh Bhatia, Sushil Kumar, Chandrakanth Are, Hareesh Nair, Klaus Nickisch, Surinder Batra. Development of cell line derived organoids to evaluate stroma targeted therapies in pancreatic 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 5792. doi:10.1158/1538-7445.AM2017-5792
Abstract 5793: Comparison of EMT biomarker expression in 2D monolayer and 3D spheroid cultures in a prostate cancer cell model
Authors: Carlstrom Jen , Hinterneder Jeanine M. , Nelson Lindsay , Hurt Stephen
Published: July 2017

The purpose of this study was to examine the induction of epithelial to mesenchymal transition (EMT) in a prostate cancer cell line by measuring classical biomarker expression in three-dimensional (3D) spheroid cultures compared to traditional 2D monolayers in an effort to develop a more biologically relevant assay. Using Ultra-Low Attachment (ULA) microplates, we grew spheroids from a human prostate cancer cell line (DU 145). Numerous studies have implicated a role for EMT in carcinoma invasion and metastasis. EMT is characterized by rearrangement of the extracellular matrix (ECM) and differential regulation of ECM proteins. We induced EMT using TGF-beta and phorbol-12-myristate-13-acetate (PMA) and compared expression levels of specific biomarkers, such as E-cadherin, fibronectin, and IL-6, using AlphaLISA and LANCE (TR-FRET) assay technologies. We confirmed that treatment of DU 145 cells with TGF-beta is sufficient for inducing changes in both EMT biomarker expression and characteristic cellular morphology in monolayer cultures. However, in 3D spheroid cultures, we observed only a partial EMT response to the same TGF-beta treatment as demonstrated by changes in the expected biomarker expression pattern. Using the small molecule, PMA, we see significant differences in the levels of IL-6 secretion after EMT induction between cells grown in monolayer and those grown in spheroids. Cellular proliferation, growth and vitality were assessed using ATPlite luminescence assays and confocal microscopy of live-stained cells with a high content imaging system. Though we observe increased proliferation in monolayer cultures compared to 3D spheroids, the changes observed in protein expression patterns cannot be sufficiently explained by differences in cell number or viability. These data illustrate the differences in protein expression levels and in cellular tolerance for compound treatment between a human prostate cancer cell line grown in monolayers and those same cells grown in 3D spheroids.

Citation Format: Jen Carlstrom, Jeanine M. Hinterneder, Lindsay Nelson, Stephen Hurt. Comparison of EMT biomarker expression in 2D monolayer and 3D spheroid cultures in a prostate cancer cell model [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 5793. doi:10.1158/1538-7445.AM2017-5793
Abstract 5812: MIEN1 promotes breast cancer cell migration and invasion by regulating cytoskeletal dynamics via focal adhesion kinase and N-WASP
Authors: Chaudhary Pankaj , Kpetemey Marilyne , Treuren Timothy Van , Vishwanatha Jamboor K.
Published: July 2017

Short Description: Migration and invasion enhancer 1 (MIEN1) is an important regulator of cell migration and invasion. MIEN1 overexpression represents an oncogenic event that promotes tumor cell dissemination and metastasis. The underlying mechanism by which MIEN1 regulates migration and invasion has yet to be deciphered.

Methods: Immunofluorescence, actin polymerization, Immunoprecipitation, migration, invasion, and adhesion assays were performed using breast cancer cell lines, MDA-MB-231 and MCF10CA1a to investigate the mechanisms by which MIEN1 potentiates cell motility.

Results: Our results show that MIEN1 acts as a cytoskeletal-signaling adapter protein to drive breast cancer cell migration. Knockdown of MIEN1 led to the loss of actin-protrusive structures whereas the overexpression of MIEN1 resulted in rich and thick membrane extensions. Knockdown of MIEN1 also decreased the cell-substratum adhesion, suggesting a role for MIEN1 in actin cytoskeletal dynamics. Our results show that MIEN1 supports the transition of G-actin to F-actin polymerization and stabilizes F-actin polymers. Additionally, MIEN1 promotes cellular adhesion and actin dynamics by inducing phosphorylation of FAK at Tyr-925 and reducing phosphorylation of cofilin at Ser-3, which results in breast cancer cell migration. Furthermore, we found that MIEN1 associates with cytoskeletal proteins involved in motility processes.

Conclusion: Our results show that MIEN1 plays an essential role in maintaining the plasticity of the dynamic membrane-associated actin cytoskeleton, which leads to an increase in cell motility. Hence, targeting MIEN1 might represent a promising means to prevent breast tumor metastasis.

Note: This abstract was not presented at the meeting.

Citation Format: Pankaj Chaudhary, Marilyne Kpetemey, Timothy Van Treuren, Jamboor K. Vishwanatha. MIEN1 promotes breast cancer cell migration and invasion by regulating cytoskeletal dynamics via focal adhesion kinase and N-WASP [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 5812. doi:10.1158/1538-7445.AM2017-5812
Abstract 5813: Rab GTPase 3C promotes colon cancer metastasis via regulation of IL-6 secretion and STAT3 signaling pathway
Authors: Chen Chi-Long
Published: July 2017

RAB GTPases is involved in membrane trafficking vesicle formation and cell motility. Recently, exocytotic RABs received growing attention in cancer research. However, the molecular mechanism of exocytotic RABs in colorectal tumorigenicity remains unclear. In this study, we found RAB GTPase 3C (RAB3C) has the most significant association with higher pathological stage, tumor recurrence, frequent distant metastasis, and poor prognosis in 215 colorectal adenocarcinoma cohort using tissue microarray (TMA) analysis. In multivariate analyses, high RAB3C expression retained its independently prognostic significance for both overall survival (p=0.001) and disease-free survival (p < 0.001). Further experimental results showed increased migration and invasion ability in RAB3C overexpressing colon cancer cells compared with control group in vitro and in vivo. The cell culture medium collected from RAB3C overexpressing model could promote parental colon cancer cells further indicated that the metastasis-promoting role of RAB3C is exocytosis dependent. We then integrated the results from previously microarray and proteomics datasets and discovered that increased production of multiple cytokines in RAB3C overexpressing cell lines. Of these cytokines, IL-6 pathway is the top pathway corresponding to gene expression changes after RAB3C overexpression. Blocking of IL-6 function with IL-6 antibody or IL-6 knockdown significantly reduced the migration ability in RAB3C overexpressing colon cancer cells. Furthermore, secretion of IL-6 after RAB3C overexpression promoted the JAK2-STAT3 signaling with increased STAT3 phosphorylation levels to promote migration. In addition, Ruxolitinib, a JAK2 inhibitor, was found to significantly inhibited RAB3C induced colon cancer cell migration through blocking STAT3 phosphorylation. In conclusion, our study showed that RAB3C overexpression promoted tumor metastasis and is associated with poor prognosis of colon cancer patients through modulating the IL6 exocytosis of cancer cells to activate JAK2-STAT3 pathway. These findings suggest a new therapeutic strategy through the inhibition of STAT3 signaling pathway in RAB3C-IL-6 axis by Ruxolitinib to prevent metastasis of colon cancer.

Note: This abstract was not presented at the meeting.

Citation Format: Chi-Long Chen. Rab GTPase 3C promotes colon cancer metastasis via regulation of IL-6 secretion and STAT3 signaling pathway [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 5813. doi:10.1158/1538-7445.AM2017-5813
Abstract 5827: Role of Ewing sarcoma EWS/FLI1 protein in midzone formation during mitosis
Authors: Azuma Mizuki
Published: July 2017

Ewing sarcoma remains the second most common form of bone and cartilage cancer in children. Although EWS/FLI1 fusion protein (derived by chromosomal translocation) is known to induce aberrant transcription in the patient cells, why and how this molecule leads to tumorigenesis specifically in skeletal cell lineage is not understood. We previously reported a novel role of EWS/FLI1, which interacts with EWS and leads to mitotic defects through an inhibition of EWS function. We further demonstrated that EWS interacts with Aurora B and relocates from inner centromere to the midzone (a midline structure that is consisted spindles called the central spindle) during anaphase of mitosis. Disruption of EWS-directed Aurora B-relocation leads to defects in cytokinesis, and to induction of chromosome instability (CIN).

EWS is an abundant molecule that is localized throughout the entire cytoplasm of anaphase, but how EWS specifically regulates midzone formation is not understood. A potential candidate mechanism is post-translational modification of EWS to modulate its activity in a spatial-temporal manner. Because previous studies showed that EWS/FLI1-T79 is a potential modification site of O-GlcNAcylation and phosphorylation, we hypothesized that the O-GlcNAcylation of EWS/FLI1 contributes to the impairment of Aurora B-relocation. Our study shows that high cellular O-GlcNAc increases the level of O-GlcNAcylation of EWS/FLI1 accompanied by the impairment of the Aurora B relocation and of central spindle formation. Contrary, EWS/FLI1-T79A and EWS/FLI1-T79D (phospho-mimic mutant) does not impair the mislocation of Aurora B at the midzone, suggesting that the O-GlcNAcylation of EWS/FLI1 is responsible for mislocation of Aurora B at the midzone. The O-GlcNAc-dependent impairment of midzone formation by EWS/FLI1 may lead to the identification of therapeutic targets for Ewing sarcoma patients.

Citation Format: Mizuki Azuma. Role of Ewing sarcoma EWS/FLI1 protein in midzone formation during mitosis [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 5827. doi:10.1158/1538-7445.AM2017-5827
Abstract 5832: Rho-associated kinase is a therapeutic target in neuroblastoma
Authors: Dyberg Cecilia H. , Fransson Susanne , Andonova Teodora , Sveinbjörnsson Baldur , Lännerholm-Palm Jessika , Forsberg David , Herlenius Eric , Martinsson Tommy , Kogner Per , Johnsen John Inge , Wickström Malin
Published: July 2017

Background: Neuroblastoma is a peripheral neural system tumor that originates from the neural crest and is the most common and deadly tumor of infancy. The non-canonical Wnt/planar cell polarity (PCP) signaling pathway regulates cytoskeletal organization, migration and maturation of neural crest cells during neuritogenesis. Here we show that neuroblastoma harbors frequent mutations of genes controlling the PCP signaling pathway and Rac/Rho signaling cascade important for proper migration and differentiation of neural crest cells during neuritogenesis. The majority of these mutations were detected in GTPase activating proteins and guanine nucleotide exchange factors genes, which may result in inhibition of Rac and activation of Rho and was associated with aggressive high-stage neuroblastomas.

Methods: We performed whole-exome sequencing of neuroblastoma samples of all clinical subgroups to search for mutations and genetic aberrations. Cytotoxic activity of Rho-associated kinases (ROCK) inhibitors was studied in cell viability assays. Morphology and invasion were studied with microscopy. The molecular mechanisms were characterized using cell- and molecular biology techniques. In vivo studies (xenografts and the transgenic mouse model TH-MYCN) in mice were carried out to validate the therapeutic effects and toxicity.

Results: Exome sequencing detected frequent mutations and gene aberrations in genes controlling the activity of PCP signaling. Analysis of gene signatures and immunohistochemistry showed that high expression of ROCK1 and ROCK2 correlated with poor patient survival. Several mediators of Rac/Rho were differentially expressed in cell lines and patient samples. Pharmacological or genetic inhibition of ROCK, a key molecule in Rac/Rho signaling resulted in differentiation, inhibition of neuroblastoma cell growth and migration and degradation of MYCN protein. Finally, ROCK inhibition reduced the growth of established neuroblastoma xenografts in nude mice and repressed tumor progression in a MYCN-driven mouse model of neuroblastoma (T-MYCN)

Conclusions: These results provide evidence that inhibition of the non-canonical/PCP signaling cascade using ROCK inhibitors suppress the growth of neuroblastoma in preclinical models and suggest possibilities for improved treatment of high-risk neuroblastomas.

Citation Format: Cecilia H. Dyberg, Susanne Fransson, Teodora Andonova, Baldur Sveinbjörnsson, Jessika Lännerholm-Palm, David Forsberg, Eric Herlenius, Tommy Martinsson, Per Kogner, John Inge Johnsen, Malin Wickström. Rho-associated kinase is a therapeutic target in neuroblastoma [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 5832. doi:10.1158/1538-7445.AM2017-5832
Abstract 584: From code to cure: Computational discovery of novel immune checkpoints
Authors: Benita Yair , Novik Amit , Cojocaru Gady , Borukhov Itamar , Wool Assaf , Kliger Yossef , Zekharya Tomer , Levine Zurit , Nemzer Sergey , Levy Ofer , Toporik Amir
Published: July 2017

Antibody blockade of CTLA4 and PD-1 immune checkpoints emerged as an effective treatment modality for cancer. However, the majority of patients do not achieve sustained long term benefit, suggesting a need for targeting of additional immune checkpoints. To identify additional B7/CD28 immune checkpoint targets, we developed a unique compendium of computational algorithms that identified multiple novel targets including TIGIT in 2008, which was an unknown protein at the time of discovery [Proc Natl Acad Sci U S A. 2009 Oct 20;106(42):17858-63], and PVRIG which we recently disclosed. Since their initial discovery, these targets have been functionally validated and anti-tumor activity was demonstrated with antibodies that target them.

In this presentation, we will describe the computational algorithms that led to the discovery of these novel immune checkpoints. These algorithms combine two complementary aspects: (i) endogenous immune checkpoint function prediction and (ii) prediction of immuno-modulatory activity in cancer. Immune checkpoint function was predicted based on gene structure similarity to B7/CD28 family members that is reminiscent of ancient common evolutionary origins. A gene structure alignment tool was developed to identify functional homologs of B7/CD28 genes even in the absence of sequence similarity. Next, the expression profile of these candidates was modeled and compared to profiles of known immune checkpoints in normal and cancer tissues. We will review the details of TIGIT and PVRIG discovery, which were among the immune checkpoints predicted in this process.

Our approach demonstrates the powerful ability of computational biology to translate genomic knowledge into rational and reliable drug target discovery.

Citation Format: Yair Benita, Amit Novik, Gady Cojocaru, Itamar Borukhov, Assaf Wool, Yossef Kliger, Tomer Zekharya, Zurit Levine, Sergey Nemzer, Ofer Levy, Amir Toporik. From code to cure: Computational discovery of novel immune checkpoints [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 584. doi:10.1158/1538-7445.AM2017-584
Abstract 5842: Therapeutic enhancement of aminolevulinic acid-mediate tumor fluorescence and photodynamic therapy with kinase inhibitors
Authors: Chen Bin , Palasuberniam Pratheeba
Published: July 2017

Photodynamic therapy (PDT) involves the combination of a photosensitizer and light of a specific wavelength. Upon light activation in the presence of oxygen, photosensitizer molecules generate reactive oxygen species that cause cytotoxicity by inducing oxidative stress. Aminolevulinic acid (ALA) is a pro-drug used for the diagnosis and PDT treatment of various solid tumors based on endogenous production of heme precursor protoporphyrin IX (PpIX). Although all human cells express heme biosynthesis enzymes and produce PpIX, tumor cells are often found to have higher PpIX production and accumulation than normal cells, allowing for the detection and treatment of solid tumors. In exploring ALA-based tumor detection and therapy in breast cancer, we have reported that high ABCG2 transporter activity in triple negative breast cancer cells (TNBC) contributes to reduced PpIX levels in tumor cells, causing cell resistance to ALA-PDT. The administration of an ABCG2 inhibitor Ko143 was able to reverse cell resistance to ALA-PDT by increasing PpIX levels. In this study, we showed that some kinase inhibitors were able to increase ALA-mediated PpIX by inhibiting ABCG2 activity. Particularly, our results demonstrate that lapatinib, a dual inhibitor of EGFR and Her2, increased the ALA-PpIX level and cell sensitivity to ALA-PDT in the MDA-MB-231 TNBC cells. Lapatinib treatment had little effect on MCF10A breast epithelial cells. These results indicate that small molecule kinase inhibitors such as lapatinib can be used for enhancing ALA-based tumor detection and PDT in tumors with high ABCG2 activity.

Citation Format: Bin Chen, Pratheeba Palasuberniam. Therapeutic enhancement of aminolevulinic acid-mediate tumor fluorescence and photodynamic therapy with kinase inhibitors [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 5842. doi:10.1158/1538-7445.AM2017-5842
Abstract 5843: Role of CDK9 inhibition as a sensitizer to radiation in esophageal adenocarcinoma: In vitro and in vivo efficacy study
Authors: Veeranki Omkara Lakshmi M. , Dokey Rashmi , Mejia Alicia , Tong Zhimin , Zhang Jianhu , Qiao Yawei , Singh Pankaj Kumar , Katkhuda Riham , Minoo Barbara , Canales Jaime Rodriguez , Lin Steven , Krishnan Sunil , Maru Dipen
Published: July 2017

Preoperative chemoradiation in neoadjuvant setting is the standard of care for loco-regional esophageal adenocarcinoma (EAC). However, less than 30% of patients develop complete pathological response indicating need for newer therapeutic strategies. Cyclin dependent kinase 9 (CDK9) is found to be over expressed in EAC compared to Barrett’s esophagus. Our previous studies demonstrated strong antitumor effects on inhibition of CDK9 in EAC both in vitro and in vivo. Here we report augmented tumor regression in irradiated xenografts on combination with Flavopiridol, a well-established clinically used CDK inhibitor, compared to single modality treatments. In vitro studies indicate that Flavopiridol could radiosensitize FLO-1 and SKGT4 EAC cells with sustained 53BP1 foci especially in SKGT4 cells. Flavopiridol and BAY1143572, a more selective inhibitor of CDK9, could radiosensitize additional EAC radiation sensitive and resistant cell lines (SKGT4-R, OE-33 and OE-33-R) as analyzed by MTT assay, apoptosis, formation of 53BP1 foci and clonogenic assay. Flavopiridol and BAY1143572 showed a radio-synergistic action by downregulating MCL-1 and Axl. In conclusion, the radio-sensitizing capacities of CDK9 inhibitors presented here suggest that their adjuvant administration might improve EAC therapy.

Note: This abstract was not presented at the meeting.

Citation Format: Omkara Lakshmi M. Veeranki, Rashmi Dokey, Alicia Mejia, Zhimin Tong, Jianhu Zhang, Yawei Qiao, Pankaj Kumar Singh, Riham Katkhuda, Barbara Minoo, Jaime Rodriguez Canales, Steven Lin, Sunil Krishnan, Dipen Maru. Role of CDK9 inhibition as a sensitizer to radiation in esophageal adenocarcinoma: In vitro and in vivo efficacy study [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 5843. doi:10.1158/1538-7445.AM2017-5843
Abstract 5858: MicroRNA-338-5p regulates radioresistance by directly targeting survivin in esophageal squamous cell carcinoma
Authors: Park Misun , Yoon Hyeon-joon , Kang Moon Chul , Lee Jun Ah , Kwon Junhye , Lee Haewon
Published: July 2017

Radioresistance is a challenge in the treatment of esophageal squamous cell carcinoma (ESCC). MicroRNAs (miRNAs) are known to play an important role in the functional modification of cancer cells and resent studies have reported the miRNA-mediated radiotherapy resistance. However, further research would be necessary to reveal the regulation mechanisms and the treatment strategies using miRNA have yet to be established in ESCC. We compared the miRNA expression profile in ESCC parental (TE-4) and acquired radioresistance (TE-4R) cell line using miRNA microarray and qRT-PCR. Our data showed that miR-338-5p, one of the target miRNA biomarkers, was significantly downregulated in TE-4R. Ectopic overexpression of the miR-338-5p induced apoptosis and sensitivity to radiation treatment by interfering the survivin, which is known to play an inhibitor of apoptosis. Over expression of survivin using plasmid vector reversed miR-338-5p induced apoptosis. From tumor xenograft experiments, we found that therapeutic delivery of the miR-338-5p mimics via direct injection to tumor mass increased sensitivity to radiation therapy. In conclusion, these findings suggested that miR-338-5p is a potential radiosensitizer and may be one of the therapeutic biomarkers for radioresistant in ESCC.

Citation Format: Misun Park, Hyeon-joon Yoon, Moon Chul Kang, Jun Ah Lee, Junhye Kwon, Haewon Lee. MicroRNA-338-5p regulates radioresistance by directly targeting survivin 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 5858. doi:10.1158/1538-7445.AM2017-5858
Abstract 5867: Reversal of drug resistance in prostate cancer using PBM nanoparticle
Authors: Singh Santosh K. , Lillard James W. , Singh Rajesh
Published: July 2017

Multidrug-resistance (MDR) is the major reason behind the failure of prostate cancer (PCa) therapy. ATP-binding cassette (ABC) transporters contribute to drug resistance via ATP-dependent drug efflux. P-glycoprotein (Pgp), which is encoded by MDR1 gene, confers resistance to certain anticancer agents. The development of agents able to modulate MDR mediated by Pgp and other ABC transporters remained a major goal for PCa therapy. The proposed study evaluated a novel method for targeted delivery of resveratrol alone or in combination with docetaxel at desired rates for reversal of drug resistance. We described a proprietary planetary ball milling approach (PBM) that uses a natural polysaccharide (starch; FDA approved) to create a drug-polysaccharide nanoparticle as a core that is subsequently coated with novel folate-conjugated poly (ϵ-caprolactone) / poly (ethylene glycol) co-polymer, which allows binding to high-affinity folate receptors presents on PCa cells. The resulting PBM nanoparticles were shown to be rapidly internalized and induce significant cancer cell apoptosis at lower doses compared to unformulated resveratrol and/or docetaxel. Our data showed that resveratrol potently synergizes with docetaxel to inhibit proliferation and induce cell death in resistance PCa cells. The up-regulation of pro-apoptotic (BAX, BID, BAK), p53 and down regulation of anti-apoptotic (BCL-2, MCL-1), Pgp and NF-κB protein confirmed that a synergistic combination of resveratrol-docetaxel with targeted delivery to tumor cells could provide a more potent therapeutic effect at lower drug concentrations and improve the therapeutic index. These results suggested that the co-delivery of resveratrol, and a cytotoxic agent in a PBM nanoparticle might potentially improve the treatment of drug-resistant tumors. [This work is supported from Grant # 5SC1CA193758-03]

Citation Format: Santosh K. Singh, James W. Lillard, Rajesh Singh. Reversal of drug resistance in prostate cancer using PBM nanoparticle [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 5867. doi:10.1158/1538-7445.AM2017-5867
Abstract 5874: Cdk4/6 kinase inhibitor resistance in prostate cancer
Authors: Leeuw Renee de , Schiewer Matthew J. , McNair Christopher , Augello Michael A. , Yoshida Akihiro , Hazard Edward S. , Courtney Sean , Hardiman Gerard T. , Drake Justin , Feng Felix Y. , Tomlins Scott , Hussain Maha H. , Diehl J Alan , Kelly William K. , Knudsen Karen E.
Published: July 2017

Non-organ confined prostate cancer (PCa) is often effectively, but only transiently treated by targeting the androgen receptor (AR) signaling axis through androgen depletion strategies, often coupled with AR antagonists. Unfortunately, disease recurs within a median of 3-4 years, presenting as castration resistant PCa (CRPC), for which there are limited therapeutic options. This emphasizes the need for more efficacious drugs and a patient-tailored approach towards cancer therapy to improve disease outcome. One class of drugs currently tested clinically, Cdk4/6 kinase inhibitors, blocks phosphorylation of the retinoblastoma (RB) tumor suppressor, thereby boosting its function, and likely preventing castration resistance. As Cdk4/6 inhibitor resistance has already been reported in other cancers, some PCa patients are anticipated to develop drug resistance. Here, we created palbociclib-resistant PCa cell models by continuously culturing them in presence of the drug to unravel mechanisms of acquired resistance, and assessed them for cross-resistance to ribociclib and response to other therapeutics. While the parental PCa cell models, Cdk4/6 inhibitors efficiently induce a G1 cell cycle arrest, the resistant cell lines bypass this cell cycle checkpoint. Although loss of RB is a known mechanism for Cdk4/6i resistance, none of the models lost RB expression. Strikingly, these originally hormone-sensitive cell lines, upon developing Cdk4/6 inhibitor resistance display altered response to selected therapeutic regimens. Mechanisms of resistance, as informed by Whole Exome Sequencing and RNASeq, will be discussed.

Citation Format: Renee de Leeuw, Matthew J. Schiewer, Christopher McNair, Michael A. Augello, Akihiro Yoshida, Edward S. Hazard, Sean Courtney, Gerard T. Hardiman, Justin Drake, Felix Y. Feng, Scott Tomlins, Maha H. Hussain, J Alan Diehl, William K. Kelly, Karen E. Knudsen. Cdk4/6 kinase inhibitor resistance 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 5874. doi:10.1158/1538-7445.AM2017-5874
Abstract 5877: The functional role(s) of serum amyloid A1 (SAA1) polymorphisms in integrin-mediated cell adhesion in nasopharyngeal carcinoma (NPC) cells
Authors: Man On Ying , Lung Hong Lok
Published: July 2017

Serum amyloid A1 (SAA1) was previously identified as a tumor suppressor gene with anti-angiogenic activities in nasopharyngeal carcinoma (NPC). Three SAA1 isoforms (SAA1.1, 1.3, and 1.5) were observed with disproportionate frequencies among the NPC patients and healthy people. SAA1.1 and 1.3 are the functional isoforms to inhibit angiogenesis whereas SAA1.5 was the defective gene. Our immunohistochemical results showed that the loss of SAA1 staining in the metastatic NPC tissues was significantly associated with tumor progression. We aim to investigate the functional roles of the three SAA1 isoforms in NPC metastasis in the present study. We want to investigate whether the functional SAA1.1 and 1.3 isoforms can suppress tumor metastasis by antagonizing the integrin-FAK signaling pathway in the NPC tumor cells. The focal adhesion assay was performed by seeding the tumor cells with or without the integrin alphaVbeta3/beta5 ligand vitronectin. Both the vector-alone control and the SAA1.5-expressing NPC cells began to spread out and adhered to the bottom of the culture dish in the presence of vitronectin from 7 to 9 hours, whereas the SAA1.1 and SAA1.3-cells remained in the round-up morphology with minimal attachment. Interestingly, after 48 hours the SAA1.1 and SAA1.3-expressing cells formed adherents junction among the cells. In order to study the viability of the effects of the three SAA1 isoforms on NPC cells, MTT viability assay was performed. The results showed that the viability of the SAA1.1 and SAA1.3-expressing cells were around 50 % lower than both the vector-alone and the SAA1.5-expressing cells. It is likely that the loss of focal adhesion after seeding of the SAA1.1 and SAA1.3-expressing cells will affect the survival of NPC cells. Furthermore, we found that the presence of the recombinant SAA1.1 and SAA 1.3 proteins could reduce the number of viable NPC cells compared with the solvent control and the SAA1.5 protein. We previously reported that the SAA1 proteins can physically interact with the integrin alphaVbeta3. Taken together, we suggested that the secreted SAA1 proteins from the NPC cells could directly affect the NPC focal adhesion as well as the cell viability by blocking the integrin on the NPC cell surface. We acknowledge the financial support of the General Research Fund (grant number HKBU17115114 to HLL) of the Research Grants Council of the Hong Kong Special Administrative Region.

Citation Format: On Ying Man, Hong Lok Lung. The functional role(s) of serum amyloid A1 (SAA1) polymorphisms in integrin-mediated cell adhesion in nasopharyngeal carcinoma (NPC) 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 5877. doi:10.1158/1538-7445.AM2017-5877
Abstract 5880: Acquired chemotherapeutic drug resistance in colorectal cancer is regulated by epithelial-to-mesenchymal transition and altered cellular pathways
Authors: Gangoda Lahiru , Mathew Nidhi , Liem Michael , Keertikumar Shiva , Ang Ching-Seng , Mariadason John , Mathivanan Suresh
Published: July 2017

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related death in the western world. Chemotherapy is the mainstay in the treatment of metastasized CRC. However, cancer cells acquire resistance to treatment by various mechanisms resulting in treatment failure. Even though the molecular mechanisms regulating acquired drug resistance is critical to overcome chemoresistance, it is poorly understood. We developed a panel of seven CRC cells resistant to 5-FU. The parental and 5-FU resistant CRC cells were assayed for proteins known for their involvement in chemotherapeutic resistance. In addition, an unbiased quantitative proteomics and DNA methylation analysis was performed on the panel of seven parental and 5-FU resistant CRC cells. The integrated analysis revealed multiple mechanisms contributing to chemotherapeutic drug resistance including epithelial-to-mesenchymal transition (EMT), deregulation of apoptosis, increased survival autophagy and epigenetic modifications resulting in altered drug metabolite potency. Inhibitors of EMT and autophagy sensitized the 5-FU resistant CRC cells. Furthermore, CRIPSR based gene knockouts of these candidate genes (both up and downregulated) either sensitized the CRC cells or rendered them resistant to 5-FU. As a follow up, PDX mouse models were established and made resistant to 5-FU. Follow up quantitative proteomics and biochemical validations of 5-FU resistant PDX tissue lysates confirmed the role of EMT in acquired chemoresistance. Overall, this project unravelled multiple mechanisms by which CRC cells may become resistant to 5-FU. Importantly, some of these mechanisms are also conserved in many cancer types and hence targeting these mechanisms can overcome chemoresistance and increase patient survival rates.

Citation Format: Lahiru Gangoda, Nidhi Mathew, Michael Liem, Shiva Keertikumar, Ching-Seng Ang, John Mariadason, Suresh Mathivanan. Acquired chemotherapeutic drug resistance in colorectal cancer is regulated by epithelial-to-mesenchymal transition and altered cellular pathways [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 5880. doi:10.1158/1538-7445.AM2017-5880
Abstract 5892: Induction of stromal fibrosis accelerates tumorigenesis in NeuT mice
Authors: Glazer Robert I. , Yuan Hongyan , Jin Lu
Published: July 2017

Background: One of the central challenges in cancer prevention is the identification of factors in the tumor microenvironment (TME) that increase breast cancer susceptibility. The stromal composition of the breast is largely adipose and fibroblast tissue, and thus it is important to understand how alterations in these constituents affect in the onset of stromal fibrosis (SF) and cancer progression. To address these questions, MMTV-NeuT mice were crossed into the FAT-ATTAC (fat apoptosis through targeted activation of caspase 8) background (NeuT/ATTAC), wherein conditional dimerization of the FKBP-caspase 8 fusion transgene in female mice results in mammary fat ablation and its replacement with fibrotic tissue.

Results: Induction of mammary fat ablation in NeuT/ATTAC mice over 4 weeks resulted in increased ductal proliferation and SF as denoted by increased collagen deposition and expression of Fibroblast Activation Protein and α-smooth muscle actin. Maintaining SF over 3-5 months resulted in a 40% reduction in tumor onset and a 2.5-fold increase in tumor multiplicity, which was associated with increased infiltration by myeloid-derived suppressor cells. Gene expression profiling of tumors from mice with and without SF identified increased expression of the immune-related genes Cxcl1, Ly6d and CD14, as well as acute phase proteins Saa1 and S100a6 in NeuT/ATTAC mice with SF.

Conclusion: Induction of SF in an erbB2-dependent breast cancer model resulted in a tumor microenvironment more conducive to tumor progression and immune tolerance. These results suggest alternative therapeutic approaches that could be taken, eg. chemokine receptor antagonists, to increase the effectiveness of immunotherapy and chemotherapy.

Supported by a grant from the Avon Foundation for Women and Award 1P30CA051008 from the National Cancer Institute, NIH, to the Lombardi Comprehensive Cancer Center.

Citation Format: Robert I. Glazer, Hongyan Yuan, Lu Jin. Induction of stromal fibrosis accelerates tumorigenesis in NeuT mice [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 5892. doi:10.1158/1538-7445.AM2017-5892
Abstract 59: Engineering glypican-3 targeting immunotoxins for the treatment of liver cancer
Authors: Fleming Bryan D. , Nixon Brittany , Pastan Ira , Ho Mitchell
Published: July 2017

The expression of glypican-3 (GPC3) in hepatocellular carcinoma offers a target with high tissue specificity and cell signaling implications. A human single domain antibody discovered by phage display technology, HN3, was fused to domain II and domain III of Pseudomonas exotoxin A. This protein (HN3-PE38) showed a high level of cytotoxic activity with a IC50 of 0.4 nM on Hep3B cells, but a relatively low maximum tolerated dose of 0.8 mg/kg in mice. In order to produce an immunotoxin with reduced toxicity, a new version was constructed that removed domain II and seven B cell epitopes from the Pseudomonas toxin. This deimmunized immunotoxin (HN3-mPE24) was shown to have a similar IC50 of 0.2 nM on Hep3B cells. To determine if further deimmunization was possible, three new versions have been generated with T cell epitopes or a series of both B and T cell epitopes removed. These include the HN3-T20 immunotoxin which had six T cell epitopes removed, HN3-T19 with 4 B cell, 4 T cell and 2 shared epitopes, and HN3-M11 with 5 B cell, 4 T cell and 2 shared epitopes. A comparative analysis of these immunotoxins was made using in vitro cell proliferation assays using Hep3B. Both the HN3-T20 (IC50 = 0.6 nM) and HN3-T19 (IC50 = 0.8 nM) immunotoxins had similar activity to HN3-mPE24 (IC50 = 0.7 nM) in a side by side comparison. The HN3-M11 variant had poor cytotoxicity and was excluded from in vivo examination. A Hep3B subcutaneous xenograft model was generated in athymic nude mice and was followed by nine rounds of intravenous immunotoxin treatments. The mPE24, T20 and T19 immunotoxins all showed an increase in average survival rate 70 days (mPE24), 76 days (T20) and 72 days (T19) when compared to the 55 days for PE38 and the 41 days for PBS alone. Additionally, the T20 and the T19 showed a maximum tolerated dose that was similar to that of the mPE24 with dosages as high as 10 mg/kg being well tolerated. This data would suggest that the HN3-T19 immunotoxins has potential clinical applications because it represents the most deimmunized immunotoxin to date.

Citation Format: Bryan D. Fleming, Brittany Nixon, Ira Pastan, Mitchell Ho. Engineering glypican-3 targeting immunotoxins for the treatment of liver 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 59. doi:10.1158/1538-7445.AM2017-59
Abstract 5904: Stiffness of extracellular matrix regulates breast cancer progression by stimulating mesenchymal stem cells
Authors: Ishihara Seiichiro , Inman David R. , Li Wan-Ju , Ponik Suzanne M. , Keely Patricia J.
Published: July 2017

The tumor microenvironment contains cancer cells, non-cancerous cells, and extracellular components such as extracellular matrix (ECM). Previous studies have demonstrated that interactions between the cells and the microenvironment contribute to cancer progression via chemical stimuli, such as growth factors, and mechanical stimuli, such as stiffness of the ECM. Recently, it has been reported that mesenchymal stem cells (MSCs) differentiate into cancer associated fibroblasts (CAFs) in response to chemical stimuli from cancer cells and thereby promote cancer progression. However, the contribution of mechanical stimuli to MSCs in cancer is poorly understood. In this study, we revealed that MSCs showed CAF phenotypes and promote mammary cancer progression in response to mechanical stimuli. On a stiff substrate, MSCs treated with conditioned media from cancer cell culture expressed increased levels of alpha smooth muscle actin (alpha-SMA), a marker of CAFs, compared to the MSCs cultured on a soft substrate. MSCs grown on a stiff substrate displayed higher expression and activity of YAP and increased phosphorylation of myosin light chain (MLC) compared to MSCs grown on a soft substrate. In addition, knockdown of YAP by shRNA decreased the expression of alpha-SMA and phosphorylation of MLC in MSCs on a stiff substrate. Pharmacological inhibition of MLC phosphorylation by H1152 treatment also reduced expression of alpha-SMA and activity of YAP in MSCs. Cell-cell communication between MSCs and carcinoma cells was bi-directional, as conditioned medium from MSCs cultured on a stiff substrate, but not a soft substrate, increased growth of mammary carcinoma cells. The soluble factor prosaposin was highly secreted by the MSCs on a stiff substrate, and the addition of recombinant prosaposin increased proliferation and survival of mammary carcinoma cells. Furthermore, secretion of prosaposin was promoted in YAP-overexpressed MSCs on a soft substrate. Mammary carcinoma cells treated with prosaposin showed increased level of phosphorylation in Akt at T308. In addition, inhibition of phosphorylation of Akt at T308 prevented proliferation and survival in mammary carcinoma cells. These results suggest that increased stiffness of the ECM in the tumor microenvironment induces differentiation of MSCs to CAFs via YAP and actomyosin contractility, secretion of prosaposion from MSCs, and as a result, trigger progression of mammary cancer via phosphorylation of Akt at T308.

Citation Format: Seiichiro Ishihara, David R. Inman, Wan-Ju Li, Suzanne M. Ponik, Patricia J. Keely. Stiffness of extracellular matrix regulates breast cancer progression by stimulating mesenchymal stem 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 5904. doi:10.1158/1538-7445.AM2017-5904
Abstract 5909: Microfluidic vertical adhesion-suspension co-culture platform for studying cancer stem cell-fibroblast interactions
Authors: Chen Yu-Chih , Zhang Zhixiong , Cheng Yu-Heng , Yoon Euisik , Wicha Max S.
Published: July 2017

Considerable evidence suggests that cancer-stem-cells (CSCs) play critical roles in tumorigenesis and metastasis .Although surface marker expression or enzymatic activity of CSC has proven useful, variability of expression highlights the need for the development of high throughput functional CSC assays. One such approach utilizes the capability of CSC’s to form single-cell-derived spheres in suspension environments (functional CSCs). Microfluidic tools are suitable for handling and monitoring a large number of single cells in suspension as well as for examining the interaction of these cells with those in the tumor micro-environment including cancer associated fibroblasts (CAF’s).In order to accomplish this, we have developed a high-throughput (10,000-well), simple-to-use, vertical platform for CSC/CAF co-culture. The platform consists of a non-adherent substrate having 10,000 micro wells for single-cell-derived sphere formation and a porous trans well for CAF adherent co-culture. As predicted by Poisson’s distribution, approximately 30% of micro wells capture single cells when T47D breast cancer cells are loaded at a concentration of 200k/ml. After cell seeding, trans wells containing CAFs are placed onto the substrate. 0.4µm pores on the trans well allow for secretion-based cellular interaction while preventing direct CSC/CAF cell contact. This cell separation facilitates sample cell retrieval without cell cross contamination. Utilizing this device, we determined that co-culture of tumor cells with CAF’s doubled the frequency of sphere formation. Spheres derived from individual CSCs were then retrieved and dissociated for single-cell transcriptome analysis utilizing RNA barcoding followed by RNAseq. Molecular analysis revealed significant cellular heterogeneity among sphere derived cells. In addition, principal component analysis of single cell RNAseq, reveled significant differences in gene expression between mono-cultured and CAF co-cultured cells isolated from tumor spheres. These genes represent potential therapeutic targets for interfering with CSC stromal cell interactions in the tumor microenvironment. The vertical adhesion suspension co-culture platform provides a system to identify and evaluate novel CSC targeted therapeutics.

Citation Format: Yu-Chih Chen, Zhixiong Zhang, Yu-Heng Cheng, Euisik Yoon, Max S. Wicha. Microfluidic vertical adhesion-suspension co-culture platform for studying cancer stem cell-fibroblast interactions [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 5909. doi:10.1158/1538-7445.AM2017-5909