bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1978: Sarcoma invasion associated with heparan sulfate degradation
Authors: Ju Donghong , Lipovich Leonard , Kosir Mary A.
Published: August 2019

INTRODUCTION: Sarcomas are locally aggressive, angiogenic and spread hematogenously. The purpose of this study was to quantify the effect of heparan sulfate degrading activity on invasion of K-ras-transformed sarcoma cells.

METHODS: Heparan degrading enzyme assays were performed in triplicate on lysate from Balb/c 3T3 (fibroblasts) and K234 cells (K-ras-transformed 3T3) cells (sarcoma) using an enzyme assay kit (cat# MK412;Takara). Invasion assays were performed using Corning Matrigel Invasion Chamber (cat#354480; ThermoFisher ). 7.5 x 104 cells were placed in serum-free DMEM in the upper chamber (insert), and DMEM with 10% FBS as attractant in the lower chamber. After incubation for 24 h at 37 °C, the insert was removed and cells on the undersurface of the insert were fixed with 5% glutaraldehyde for 10 min, then stained with 1% crystal violet in 2% ethanol for 20 min. After the insert was dry, the cells on the filter were counted by microscope over multiple fields. For experiments with inhibitors, 10 ug/ml λ-carageenan (cat# 22049; Sigma), anti-NAP-2 antibody (cat# sc-19224; Santa Cruz Biotech), or anti-HPA1 antibody were used.

RESULTS: Heparan sulfate-degrading activity was 0.0625 U/ug for K234 cell lysate compared to 0.0125 U/ug for Balb/c3T3 cells. λ-carageenan inhibited the activity completely confirming glycanase inhibition. Anti-NAP2 and anti-HPA1 antibody inhibited heparan sulfate degrading activity in 3T3 lysate. Invasion was two-fold greater by K234 cells than for 3T3 cells. When anti-NAP2 antibody was added to the invasion assay, both K234 and 3T3 cell invasion was inhibited by 55% and 54%, respectively. Less than 1% inhibition of invasion occurred with anti-HPA1 antibody.

CONCLUSION: The invasion of heparan sulfate-containing Matrigel by K234 cells is partly influenced by heparan sulfate-degrading activity. The inhibition of the degrading activity by an anti-chemokine antibody decreased invasion, and invokes other processes for the malignant sarcoma phenotype.

Citation Format: Donghong Ju, Leonard Lipovich, Mary A. Kosir. Sarcoma invasion associated with heparan sulfate degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1978.
Abstract 1983: Cancer exosomes immunosuppressive activity in the absence of contaminating soluble factors is multifactorial
Authors: Shu Shin La , Matsuzaki Junko , Allen Cheryl , Yang Yunchen , Hurley Edward , Odunsi Kunle , Wu Yun , Ernstoff Marc S.
Published: August 2019

Cancer derived exosomes, along with soluble factors such as growth factors, cytokines and chemokines have been identified as integral immunosuppressive pathways employed by cancer cells. Using melanoma cell lines as a model for generating human melanoma exosomes (HMEX), we studied the soluble factors associated with exosome purification methods. A Luminex 13-plex T cell activation kit was used to detect the presence of soluble factors in exosome isolates. We have demonstrated that combining Ultrafiltration and Size Exclusion Chromatography (REIUS method) can yield up to 58-fold more exosomes than ultracentrifugation (UC) with greatly reduced concentrations of contaminating soluble factors (IL-8: 4.6 pg/ml by REIUS versus 489 pg/ml by UC | IL-10: 1,052 pg/ml by REIUS versus 34,500 pg/ml by UC). A novel exosome-based Compact Surface Plasmon Resonance Biosensor demonstrated that REIUS-isolated exosomes abundantly express PD-L1 on their surface compared to UC-purified exosomes. These HMEX significantly inhibited a cloned NY-ESO tumor antigen-specific patient derived T cell function when co-cultured with a NY-ESO positive melanoma cell line for 4 h (IFN-γ positive T cell population: 78.5 ± 0.03 % versus 49.05 ± 0.07 %). Blocking with anti-PDL1 or anti-IL-10 failed to restore T cell function (IFN-γ production), while both anti-PD-L1 and anti-IL-10 antibodies partially reverse this suppression, thereby identifying IL-10 and PD-L1 as a novel collaborating inhibitory pathway. Taken together, we report that different exosome isolation methods impact the nature of the exosomes and contaminating soluble factors and should be chosen with caution. In addition, our data suggests a novel role for tumor-derived exosomes in the IL-10 and PD1-PDL1 axis crosstalk that impacts functional activity of T cells.

Citation Format: Shin La Shu, Junko Matsuzaki, Cheryl Allen, Yunchen Yang, Edward Hurley, Kunle Odunsi, Yun Wu, Marc S. Ernstoff. Cancer exosomes immunosuppressive activity in the absence of contaminating soluble factors is multifactorial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1983.
Abstract 1986: Breast cancer-secreted exosomes stimulate beige/brown differentiation and reprogram metabolism in stromal adipocytes to promote tumor progression
Authors: Li Juanjuan , Wu Qi , Li Zhiyu , Sun Si , Zhu Shan , Wang Lijun , Wu Juan , Zhang Yimin , Yuan Jingping , Sun Shengrong , Wang Changhua
Published: August 2019

Background: Emerging evidence supports the pivotal roles of adipocytes in breast cancer progression. Beige/brown adipose tissue that activates thermogenesis is suggested to contribute to the hypermetabolic state. However, the mediators and mechanisms remain unclear.

Methods: Survival probabilities for recurrence-free survival (RFS) of 106 breast cancer patients were estimated using the Kaplan-Meier method based on the immunohistochemistry results of several biomarkers. Biochemical studies were performed to characterize the novel interrelation between breast cancer cells and adipocytes.

Results: In the survival analysis, high MCT4 expression in stromal adipose tissue and overexpression of CD36 or FATP1 in malignant tissue predict poor prognosis for breast cancer patients. We show that tumor-surrounding adipocytes exhibit an altered phenotype in terms of upregulated beige/brown characteristics and increased catabolism. This increase in catabolism is associated with an activated state characterized by the release of metabolites, including free fatty acids, pyruvate, lactate and ketone bodies. Likewise, tumor cells cocultivated with mature adipocytes exhibit metabolic adaptation and an aggressive phenotype in vitro and in vivo. Mechanistically, we show that tumour cells induce beige/brown differentiation and remodel metabolism in resident adipocytes by secreting exosomes that carry high levels of miRNA-144 and miRNA-126. Cancer cell-secreted miRNA-144 promotes beige/brown adipocyte characteristics by downregulating the MAP3K8/ERK1/2/PPARγ axis. We also found that exosomal miRNA-126 remodels metabolism by disrupting IRS/Glut-4 signalling, activating the AMPK/autophagy pathway and stabilizing HIF1α expression in imminent adipocytes. In vivo inhibition of miRNA-144 or miRNA-126 decreases adipocyte-induced tumour growth.

Conclusions: These results demonstrate that cancer-derived exosomal miRNA-144 and miRNA-126 can reprogram the systemic energy metabolism to facilitate tumor progression by inducing beige/brown differentiation and enhancing catabolism in recipient adipocytes.

Citation Format: Juanjuan Li, Qi Wu, Zhiyu Li, Si Sun, Shan Zhu, Lijun Wang, Juan Wu, Yimin Zhang, Jingping Yuan, Shengrong Sun, Changhua Wang. Breast cancer-secreted exosomes stimulate beige/brown differentiation and reprogram metabolism in stromal adipocytes to promote tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1986.
Abstract 1990: Cjc-1134-pc, a long-acting glp-1r agonist, effects in modulating pro-inflammatory cytokines in the tumor environment
Authors: Liu Xiping , Hong Sean , Wu Shijuan , Yao Zhiwen , Xie Qiang
Published: August 2019

The incidence rates of The incidence rates of diabetes and cancer are growing globally. It is reported that type 2 diabetes (T2D) is associated with increased risk of cancers, which suggests a synergy. There is a strong association among inflammation and cancer on diabetes, which is reflected by the high cytokine prevalence in the microenvironment. Previously our studies showed that IL-2 and IFN-γ were downregulated but IL-6 and TNFα were upregulated in diabetic mice. The levels of IL-2, IFN-γ, IL-6 and TNFα returned to baseline levels similar to that of non-diabetic mice after the treatment of CJC-1134-PC. In order to explore the benefits of CJC-1134-PC beside its glucose lowering effects, the level of pro-inflammatory cytokines has been measured and compared between vehicle and CJC-1134-PC treated BALB/c mice bearing CT26 colon tumors. All mice bearing tumors showed high IL-6 and TNF-r compared with the normal mice. Interestingly, after CJC-1134-PC treatment, IL-6 and TNFα levels decreased significantly (P < 0.05), which makes the cytokine profile close to the reported levels in normal mice. Overall, the data suggests that CJC-1134-PC plays a role in modulating inflammation, which could benefit cancer treatment in addition to achieving glycemic control.

Citation Format: Xiping Liu, Sean Hong, Shijuan Wu, Zhiwen Yao, Qiang Xie. Cjc-1134-pc, a long-acting glp-1r agonist, effects in modulating pro-inflammatory cytokines in the tumor environment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1990.
Abstract 1993: Gluthathione peroxidase 2 downregulation in the PyMT mammary tumor model leads to an aggressive phenotype due to stimulation of angiogenesis and tumor stemness via HIF1 alpha signaling
Authors: Ren Zuen , Liang Huizhi , Benard Outhiriaradjou , Suyama Kimita , Albanese Joseph M. , Norton Larry , Zheng Wei , Hazan Rachel B.
Published: August 2019

Breast tumors are known to undergo high levels of mitochondrial oxidative phosphorylation leading to accumulation of reactive oxygen species (ROS), which in turn potentiate cancer stem cells and chemoresistance. This was shown to be due to HIF1α stabilization by ROS; however, the signaling events downstream of HIF1α regulating the cancer stem cell (CSC) phenotype remain unknown. Kaplan Meier survival curves upon the gene profiling database of breast cancer patients showed that reduced levels of the ROS scavenging enzyme, glutathione peroxidase 2 (GPx2) in breast cancers was associated with shorter patient survival duration. Consistent with this idea, we found that GPx2 was downregulated in metastatic mammary tumor cells, suggesting GPx2 loss enhances metastasis. Indeed, GPx2 knockdown in poorly metastatic cells increased ROS production, shortened tumor latency, and stimulated dramatic mammary tumor growth and metastasis. These effects were associated with marked increases in angiogenesis, CSC-like properties, and chemoresistance. Here, we show that GPx2 downregulation in mammary epithelial cells increases ROS production and HIF1α stabilization which were accompanied by VEGF upregulation and enhanced angiogenesis. On the other hand, GPx2 loss caused dramatic mammosphere formation and resistance to doxorubicin, implying effects on stemness leading to chemoresistance. These data suggest that GPx2 loss is a critical event that drives mammary tumorigenesis and metastasis via HIF1α signaling leading to stimulation of angiogenesis and stemness properties. Understanding the ramifications of GPx2 loss in breast cancer will allow us to identify key regulators of stemness and chemoresistance, with the goal of identifying novel drug targets in metastasis.

Citation Format: Zuen Ren, Huizhi Liang, Outhiriaradjou Benard, Kimita Suyama, Joseph M. Albanese, Larry Norton, Wei Zheng, Rachel B. Hazan. Gluthathione peroxidase 2 downregulation in the PyMT mammary tumor model leads to an aggressive phenotype due to stimulation of angiogenesis and tumor stemness via HIF1 alpha signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1993.
Abstract 2: Discovery of BAY 2402234 by phenotypic screening: A human Dihydroorotate Dehydrogenase (DHODH) inhibitor in clinical trials for the treatment of myeloid malignancies
Authors: Gradl Stefan N. , Mueller Thomas , Ferrara Steven , Sheikh Sherif El , Janzer Andreas , Zhou Han-Jie , Friberg Anders , Guenther Judith , Schaefer Martina , Stellfeld Timo , Eis Knut , Kroeber Michael , Nguyen Duy , Merz Claudia , Niehues Michael , Stoeckigt Detlef , Christian Sven , Zimmermann Katja , Lejeune Pascal , Bruening Michael , Meyer Hanna , Puetter Vera , Scadden David T. , Sykes David B. , Seidel Henrik , Eheim Ashley , Michels Martin , Haegebarth Andrea , Bauser Marcus
Published: August 2019

DHODH is a key enzyme in the biosynthesis of pyrimidines and recent studies have renewed interest in this old anti-cancer target. Here, we disclose the discovery of 4-triazolosalicylamides as inhibitors of DHODH and their structure activity relationship (SAR). The hit cluster was discovered during a phenotypic high throughput screen (HTS) of 2.5 million compounds where proliferation of H460 lung cancer cells was used as read-out. DHODH was successfully identified as the molecular target by comparing the activity profile of the hits in a panel of cell lines to a set of inhibitors with known pharmacological activity. The hit compounds showed good cellular potency but had undesirable DMPK properties. Interestingly, the compounds are non-ionizable in contrast to many other DHODH inhibitors and show no potency shift from biochemical to cellular assays. Structural modifications lead to compounds with sub-nanomolar potency in cellular assays and increased metabolic stability enabling the proof of concept in vivo xenograft experiments. Further optimization guided by lipophilicity efficiency and identification of metabolic hot spots resulted in molecules with low clearance and improved solubility. BAY 2402234 was selected as the clinical candidate after side by side comparison of a number of promising compounds. It shows great oral bioavailability, target engagement in all preclinical species tested, induces differentiation in AML models, and has excellent activity in a variety of leukemia models. A clinical phase I study has been initiated in patients with myeloid malignancies. (NCT03404726)

Citation Format: Stefan N. Gradl, Thomas Mueller, Steven Ferrara, Sherif El Sheikh, Andreas Janzer, Han-Jie Zhou, Anders Friberg, Judith Guenther, Martina Schaefer, Timo Stellfeld, Knut Eis, Michael Kroeber, Duy Nguyen, Claudia Merz, Michael Niehues, Detlef Stoeckigt, Sven Christian, Katja Zimmermann, Pascal Lejeune, Michael Bruening, Hanna Meyer, Vera Puetter, David T. Scadden, David B. Sykes, Henrik Seidel, Ashley Eheim, Martin Michels, Andrea Haegebarth, Marcus Bauser. Discovery of BAY 2402234 by phenotypic screening: A human Dihydroorotate Dehydrogenase (DHODH) inhibitor in clinical trials for the treatment of myeloid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2.
Abstract 2001: Inhibiting voltage-gated sodium channel activity in medullary thyroid cancer using small molecule compounds
Authors: Ahuja Paras , Guenter Rachael , Cowan Jaden , Shihab Yazen , Whitt Jason , Chen Herbert , Velu Sadanandan , Jaskula-Sztul Renata
Published: August 2019

Medullary thyroid cancer (MTC) is a type of neuroendocrine cancer (NE) with increasing incidence worldwide. Compared with well-differentiated thyroid cancers, distant metastases are more commonly observed in MTC patients, ranging from 7-23%. The inability to treat metastatic MTC contributes to a decreased patient survival rate. Currently, at least nine mammalian voltage-gated sodium channels (VGSCs) named Nav1.1-Nav1.9 have been identified as potential safe drug targets for treating metastasis in various cancers. Herein, we investigate the effects of targeting VGSCs in MTC using small molecule inhibitors. Two MTC cell lines, TT and MZ, were treated with four compounds and the changes in mRNA and protein expression of VGSC isoform Nav1.7 were assessed through RT-PCR and western blotting. Treatment by one of the compounds exhibited a 28-fold decrease in mRNA expression of VGSC isoform 1.7. Furthermore, a cell proliferation assay, proliferation assay (MTT) a MTT, assay to study cell proliferation after treatment showed decreasing cell viability resulted from the treatment. and Additionally, morphological changes characteristic of apoptosis in fluorescently labeled MTC cell lines after 48 hours of treatment with VGSC inhibitory compounds were observed. A Boyden chamber assay was also performed on MTC cell lines to examine changes in migration and invasion after administering the potential inhibitory compounds. This study has generated preliminary evidence that voltage-gated sodium channels (VGSCs) may indeed be potential targets for treating metastatic MTC. The results may be translated to future studies focused on controlling metastasis through specified drug treatment in MTC, leading to the limitation of cancer progression by means of targeting VGSCs.

Citation Format: Paras Ahuja, Rachael Guenter, Jaden Cowan, Yazen Shihab, Jason Whitt, Herbert Chen, Sadanandan Velu, Renata Jaskula-Sztul. Inhibiting voltage-gated sodium channel activity in medullary thyroid cancer using small molecule compounds [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2001.
Abstract 2014: A novel small molecule inhibitor to suppress Notch1 activation in CRC
Authors: Damodaran Chendil , Tyagi Ashish , Chandrasekaran Balaji , Kolluru Venkatesh , Jojua Ibrahim , Ramisetti Srinivasa , Sharma Arun K. , Ankem Murali K.
Published: August 2019

Introduction: Earlier we reported that aberrant overexpression of Notch1 in colon cancer patients is responsible for aggressiveness and progression of the disease. Current, therapeutic interventions of notch1 inhibitors that binds catalytic unit of γ-secretase and disrupts the cleavage activity cause gastrointestinal toxicity in clinical trial. Therefore, our study aims to develop a small molecule inhibitor, which inhibits Notch1 signaling but binds outside of γ-secretase’s catalytic unit thereby substantially reducing associated toxicity attributes. Through structure-activity relationship (SAR) studies based on naturally occurring Withaferin A (WA) structure, we have recently identified a WA-difuroate analog ASR458 as one such small molecule.

Methods: To analyze the effect of ASR490 on HCT116 and HCT116/Notch1 cells, we performed phenotypic, western blot and in vivo analysis.

Results: Notch1 overexpressing CRC cells (HCT/Notch1) showed aggressive cell growth (42%) as compared to HCT116 cells, however, ASR490 treatment inhibited cell growth in both HCT116 (IC50: 600 nM) as well as HCT/Notch1 cells (IC50: 850 nM). ASR458 significantly inhibited Notch1 expression and its downstream events in both the transfected cell lines. As a result, we observed shutdown of pro-survival machinery and induction of pro-apoptotic events (Bax, caspases and PARP) resulting in apoptotic cell death in ASR456 treated HCT116 and Notch1/HCT116 cells. Since Notch1 is responsible for epithelial mesenchyme transition (EMT); we analyzed EMT drivers and readout invasion/migration assays. ASR458 treatment reversed the mesenchymal characteristics by restoring the function of E-cadherin, which resulted in the suppression of invasion and migration in readout assays of HCT116 transfectants. Our ongoing Xenotransplanted mice experiments may suggest whether ASR458 inhibits the growth of HCT116 and HCT116/Notch1 tumors.

Conclusion: Restrained gastrointestinal toxicity of Notch1 targeting therapeutics is achievable with novel small molecules displaying a differential binding affinity to γ-secretase. ASR458 inhibits cell growth in Notch1 overexpressing cells and abrogates Notch1 induced EMT. Ongoing molecular docking and in vivo studies may confirm ASR458 binding sites in γ-secretase and validate our in vitro findings respectively.

Citation Format: Chendil Damodaran, Ashish Tyagi, Balaji Chandrasekaran, Venkatesh Kolluru, Ibrahim Jojua, Srinivasa Ramisetti, Arun K. Sharma, Murali K. Ankem. A novel small molecule inhibitor to suppress Notch1 activation in CRC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2014.
Abstract 2015: Downregulation of MZB1 by aminoguanidine, APOE mimetic and nitroxyl compounds associated with reduction in invasive breast tumor volume and metastasis
Authors: Cheng Yuk Sing Robert , Basudhar Debashree , Lisa Rdinour , Somasundaram Veena , Wink David
Published: August 2019

Triple negative breast cancer (TNBC) is notoriously known for poor prognosis due to a lack of actionable molecular target(s). Remarkably, the majority of TNBC patients suffer the cancer relapse even they receive treatment given for early stage breast cancers. Up-to-date, lots of therapeutic agents have been developed and actionable molecular targets have been proposed. Nevertheless, the survival rate for metastatic invasive breast cancer is low because of poor prognosis. In this study, we determined whether different classes of chemo-agents (or compounds) interact with different cellular and molecular pathways they might share some common pathway(s) if their therapeutic outcome is similar. We first tested the effect of aminoguanidine (AG), APOE mimetics (COG), and nitroxyl (HNO) compounds on carcinogenesis and metastasis using an orthotopic breast cancer model. In vivo imaging analysis indicated that these compounds significantly reduced the tumor loading in the xenografts (MDA-MB-231-GFP). Moreover, compounds AG and COG are able to reduce brain metastasis significantly. RNA-Seq analysis identified only one gene which is commonly altered in all treatment groups. This gene encodes marginal zone B and B1 cell specific protein (MZB1). MZB1 was downregulated after treatment and its expression was inversely correlated with the efficacy of brain metastasis. In addition, pathway comparison analysis of RNA-Seq data revealed there are three top canonical pathways including (1) Role of NFAT in Regulation of the Immune Response, (2) PKC theta Signaling in T Lymphocytes, and (3) iCOS-iCOSL Signaling in T Helper Cells, downregulated in all treatment groups. In conclusion, our findings demonstrated three different classes of compounds can reduce not only tumor loading but metastasis. MZB1, identified from our transcriptome analysis, may serve as an actionable molecular target for TNBC. All-in-all, it provides new insights into the development of new therapeutic targets and biomarkers for TNBC.

Citation Format: Yuk Sing Robert Cheng, Debashree Basudhar, Rdinour Lisa, Veena Somasundaram, David Wink. Downregulation of MZB1 by aminoguanidine, APOE mimetic and nitroxyl compounds associated with reduction in invasive breast tumor volume and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2015.
Abstract 2020: Adipocytes up-regulates CD36 expression, fatty acid uptake and oxidation enhancing breast cancer cell progression
Authors: Choi Junjeong , Gyamfi Jones
Published: August 2019

The role of the tumor microenvironment in tumor progression has recently gained enormous attention, with studies reporting heterogeneous interactions between tumor cells and tumor stroma cells driving tumor progression. The breast cancer microenvironment is unique since the breast tissue within which the tumor originates comprises predominantly of adipocytes. This makes adipocytes a unique component in the breast cancer microenvironment. Adipocytes secrete various growth factors and cytokines that influence tumor progression by induction of epithelial-mesenchymal transition (EMT) which enhances cancer cell migration, invasion and metastasis. This study presents an alternative approach by which adipocytes may enhance cancer progression via the transfer of free-fatty acids from tumor-associated adipocytes to breast cancer cells. The uptake of free fatty acid subsequently serves as a secondary source of energy to drive breast cancer progression. The study aimed to provide an in-depth understanding of the molecular mechanism by which free fatty acid are imported and metabolized in cancer cells in an adipocyte-rich microenvironment. Human adipocytes were co-cultured with cancer cells and the fatty acid (FA) translocase (CD36) activity was inhibited by the chemical inhibitor sulfosuccinimidyl oleate (SSO). Silencing RNA and expression plasmids were used to evaluate CD36 activity in cancer cells. Expression levels of key genes and proteins involved in lipid transfer and metabolism were evaluated by qtPCR and western blot. Inhibition of CD36 reduces free fatty acid uptake and induces intracellular lipogenesis with upregulation of fatty acid synthase (FASN). Breast cancer cells co-cultured with human adipocytes express high levels of the CD36 facilitating free fatty acid uptake from adipocytes. Co-cultured cells had increased levels of fatty acid oxidation (FAO) and oxygen consumption, with increased migration and invasion characteristics. We also observed a significant increase in the expression of CPT1A required for mitochondrial fatty acid import and ATGL required for triglyceride metabolism. Our findings indicate an enhanced dependence on FAO in co-cultured cancer cell. Indicating a potential bioenergetic adaptation of breast cancer cells to the adipocyte-rich microenvironment. Inhibition of CD36 and CPT1 significantly decreased breast cancer cell migration and invasion. These results suggest that cancer-associated adipocytes induce free fatty acid uptake via upregulation of CD36 and reprogram breast cancer cell metabolism. Thus, targeting CD36 may have a potential targeted therapy.

Citation Format: Junjeong Choi, Jones Gyamfi. Adipocytes up-regulates CD36 expression, fatty acid uptake and oxidation enhancing breast cancer cell progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2020.
Abstract 2021: Role of MMP1-PAR-1 crosstalk in the pro-tumorigenic senescent fibroblasts in large cell carcinoma of the lung
Authors: Gabasa Marta , Ikemori Rafael , Radisky Evette , Reguart Noemí , Radisky Derek , Alcaraz Jordi
Published: August 2019

Tumor associated fibroblasts (TAFs) are key effector cells of cancer progression. Intriguingly, senescent TAFs have been reported in a growing list of agressive cancer subtypes including large cell carcinoma (LCC) of the lung. We previously reported that LCC cells induce fibroblast senescence in indirect co-cultures with transwells, revealing that paracrine signaling must be involved. A follow-up study identified MMP1 as an important regulatory protein in fibroblast senescence, since knocking-down MMP1 in LCC cells was sufficient to abrogate the induction of fibroblast senescence in co-cultures as well as the growth and invasion enhancement elicited by the conditioned medium of fibroblasts in LCC cells. Here we examined the potential role of the protease activated protein 1 (PAR-1) in fibroblast senescence by MMP1, since PAR-1 can be activated by MMP1 and has been reported in the stroma of lung cancer patients. We found that PAR-1 expression was down-regulated in fibroblasts that become senescent upon co-culture with LCC cells. In addition, forcing a reduced PAR-1 expression in fibroblasts by siRNA increased their expression of markers of the senescence associated secretory phenotype (SASP) during co-culture with LCC cells, whereas the percentage of senescence-associated beta-galactosidase positive fibroblasts remained largely unaffected. These results suggest that fibroblast senescence elicited by MMP1 secreted by LCC cells is mediated by processes other than PAR-1 activation and/or overexpression in fibroblasts.

Citation Format: Marta Gabasa, Rafael Ikemori, Evette Radisky, Noemí Reguart, Derek Radisky, Jordi Alcaraz. Role of MMP1-PAR-1 crosstalk in the pro-tumorigenic senescent fibroblasts in large cell carcinoma of the lung [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2021.
Abstract 2028: CCL18 signaling from breast tumor-associated macrophages fosters the activation of fibroblasts into a chemoresistance-inducing phenotype
Authors: Song Erwei , Chen Jianing , Su Shicheng
Published: August 2019

Carcinoma-associated fibroblasts (CAFs) are abundant and heterogeneous stromal cells in tumor microenvironment critically involved in cancer progression. Our recent study identified a specific CAFs subset by two cell-surface molecules, CD10 and GPR77. We proved that CD10+GPR77+CAFs could promote tumor formation and chemoresistance by providing a survival niche for cancer stem cells (CSCs). However, their origin and underlying activating initiation mechanism are unclear. Tumor-associated macrophages (TAMs) are the most abundant immune-related stromal cells and the important source of inflammatory cytokine. Here, we demonstrated that CCL18 secreted by TAMs could induces the activation of normal breast fibroblasts into a CD10+GPR77+CAFs phenotype, which are not only resistant to chemotherapy themselves, but also can induce chemoresistance in cancer cells by abundantly producing IL-8 and IL-6 and enriching the population of cancer stem cells. In conclusion, our studies have identified an inflammatory signaling network in the interaction of different kind of stroma cells in tumor microenvironment and highlighted its potential values as predictive markers and therapeutic targets to abrogate tumor formation and improve chemotherapeutic efficacy.

Note: This abstract was not presented at the meeting.

Citation Format: Erwei Song, Jianing Chen, Shicheng Su. CCL18 signaling from breast tumor-associated macrophages fosters the activation of fibroblasts into a chemoresistance-inducing phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2028.
Abstract 2029: Identification of cancer-associated fibroblasts that suppress pancreatic cancer progression
Authors: Enomoto Atsushi , Mizutani Yasuyuki , Kobayashi Hiroki , Iida Tadashi , Woods Susan L. , Worthley Daniel L. , Takahashi Masahide
Published: August 2019

Cancer-associated fibroblasts (CAFs) constitute a major component of the cancer stroma. They can promote cancer progression through a variety of mechanisms including the production of growth factors, chemo/cytokines, and extracellular matrix (ECM). Recent observations from genetically engineered mouse models and clinical studies have suggested that there may exist at least two subpopulations of CAFs, i.e., cancer-promoting CAFs (pCAFs) and cancer-restraining CAFs (rCAFs). Although various pCAF markers have been identified, the identity of rCAFs is unknown due to a lack of specific rCAF marker(s). Here, we show that Meflin, a glycosylphosphatidylinositol (GPI)-anchored protein that maintains the undifferentiated state of mesenchymal stromal/stem cells (MSCs) (Maeda et al., Sci Rep, 6:22288, 2016), is a marker of pancreatic stellate cells (PSCs). We find that Meflin-positive CAFs represent rCAFs in pancreatic ductal adenocarcinoma (PDAC). Infiltration of Meflin-positive CAFs correlated with favourable prognosis in patients with PDAC, consistent with our observation that Meflin deficiency led to tumour progression with poorly differentiated histology in a PDAC mouse model. Notably, Meflin-positive cells gave rise to α-smooth muscle actin (SMA)-positive CAFs during cancer progression. Both genetic ablation of Meflin-positive CAFs and delivery of a Meflin-expressing lentivirus into the stroma suppressed xenografted tumour differentiation and growth, respectively. Mechanistically, Meflin interacts with lysyl oxidase (Lox) to inhibit collagen crosslinking activity, and Meflin deficiency led to straightened and wide stromal collagen fibers as demonstrated by a second harmonic generation microscopy. These data demonstrate the presence of rCAFs in the cancer stroma and the significance of their differentiation to pCAFs in cancer progression, which may be exploited for the development of therapeutic strategies to specifically target pCAFs or reprogram them into rCAFs.

Citation Format: Atsushi Enomoto, Yasuyuki Mizutani, Hiroki Kobayashi, Tadashi Iida, Susan L. Woods, Daniel L. Worthley, Masahide Takahashi. Identification of cancer-associated fibroblasts that suppress pancreatic cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2029.
Abstract 2037: Macrophage Inflammatory Protein-3 beta (MIP-3β) from cancer-associated fibroblasts promote the proliferation of melanoma cells
Authors: Fujikawa Heishiro , Yashiro Masakazu , Hatano Takaharu , Maeda Shusaku , Motomura Hisashi
Published: August 2019

Introduction: Malignant tumors are composed of cancer cells and various types of stromal cells, including fibroblasts, vascular endothelial cells, and immune cells. It has been reported that fibroblasts in the tumor stroma termed as cancer-associated fibroblasts (CAFs) may affect cancer progression. However, the currently available evidence regarding the function of CAFs remains controversial, with some studies reporting stimulatory activity and some studies reporting inhibitory activity in malignant melanoma cells. Previously, we reported that CAFs possibly exert a stimulatory effect on the proliferation of melanoma cells. In this study, we identified a factor from CAFs that are involved in melanoma progression.

Materials and Methods: We used three malignant melanoma cell lines, namely MMAc, COLO679, and A375. Three CAFs and one type of non-affected fibroblasts (NAFs) were established from sebaceous carcinoma/skin malignant melanoma and normal skin, respectively. Serum-free conditioned medium from CAFs (CAF-CM) was prepared as follows: CAFs were seeded into 100-mm plastic dishes with 10 ml of Dulbecco's modification of Eagle medium (DMEM) containing 5% fetal calf serum. To obtain CAF-CM, CAFs were washed using phosphate-buffered saline and subsequently incubated for 3 days with serum-free DMEM, and the conditioned medium was collected as CAF-CM. Additionally, we obtained NAF-CM from NAFs using the same procedure. These fibroblasts were cultured and used before the 15th passage. The cytokines included in each conditioned medium were determined using cytokine assay. Subsequently, we selected some cytokines, which were released more abundantly in CAFs than in NAFs, as potential factors affecting the proliferation of melanoma cells. The effects of these cytokines on the proliferation of melanoma cells were determined using MTT assay and cytometry assay using a Coulter Counter.

Results: CAF-CM contained significantly high concentration of MIP-3β, in compared with that in NAF-CM. In addition, MIP-3β significantly increases in the proliferation of melanoma cells, A375 and COLO679, at 48 h and 72 h culture.

Conclusion: CAFs from skin cancer might promote the proliferation of malignant melanoma cells via MIP-3β signaling.

Citation Format: Heishiro Fujikawa, Masakazu Yashiro, Takaharu Hatano, Shusaku Maeda, Hisashi Motomura. Macrophage Inflammatory Protein-3 beta (MIP-3β) from cancer-associated fibroblasts promote the proliferation of melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2037.
Abstract 2056: Combined FLT3 and Pim kinase inhibitor treatment downregulates c-Myc early in apoptosis induction in acute myeloid leukemia with FLT3-ITD
Authors: Lee Jonelle K. , Scarpa Mario , Kapoor Shivani , Baer Maria R.
Published: August 2019

Background: fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is present in acute myeloid leukemia (AML) in 30 % of patients, with poor treatment outcomes due to rapid relapse. FLT3 inhibitors have limited activity and rapid onset of resistance. The oncogenic serine/threonine kinase proviral integration site for Moloney murine leukemia virus 1 (Pim-1) is upregulated downstream of FLT3-ITD and phosphorylates and stabilizes FLT3 in a positive feedback loop in cells with FLT3-ITD. Dual inhibition of FLT3 and Pim kinases is therefore a promising treatment strategy for AML with FLT3-ITD. The proto-oncogene c-Myc, upregulated in many cancer types, drives proliferation and causes resistance to apoptosis. We previously showed that concurrent FLT3 and Pim inhibition in cells with FLT3-ITD increases apoptosis through downregulation of the anti-apoptotic protein Mcl-1. Here we demonstrate downregulation of c-Myc upstream of Mcl-1.

Methods: Ba/F3-ITD and MV4-11 cells, with FLT3-ITD, were cultured with the FLT3 inhibitors gilteritinib (15 nM) or quizartinib (1 nM) and the pan-Pim inhibitor AZD1208 (1 μM) alone and in combination, and with DMSO control. c-Myc and Mcl-1 protein expression was measured by immunoblotting and c-Myc mRNA by qPCR using SybrGreen. Cells were cultured with cycloheximide (100 μg/mL) with and without the proteasome inhibitor MG-132 (20 μM). Ba/F3-ITD cells were stably transfected with a lentiviral c-Myc plasmid or a retroviral Mcl-1 plasmid and corresponding empty vectors. Apoptosis was detected by Annexin V and propidium iodine staining, measured by flow cytometry.

Results: Combined FLT3 and Pim inhibitor treatment decreased c-Myc protein expression in Ba/F3-ITD and MV4-11 cells within 30 minutes, preceding Mcl-1 downregulation. c-Myc mRNA levels did not change, but c-Myc protein half-life decreased with combination treatment, and this decrease was abrogated by proteasome inhibitionMcl-1 overexpression did not abrogate c-Myc downregulation by combination treatment, and c-Myc overexpression decreased apoptosis induction by combination treatment from 65% to 45%.

Conclusion: Combined FLT3 and Pim kinase inhibitor treatment enhances apoptosis in FLT3-ITD cells through c-Myc downregulation, which precedes Mcl-1 downregulation. c-Myc downregulation is post-translational, via increased proteasomal degradation.

Citation Format: Jonelle K. Lee, Mario Scarpa, Shivani Kapoor, Maria R. Baer. Combined FLT3 and Pim kinase inhibitor treatment downregulates c-Myc early in apoptosis induction in acute myeloid leukemia with FLT3-ITD [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2056.
Abstract 2058: BCL-2 selective inhibitor APG-2575 synergizes with BTK inhibitor in preclinical xenograft models of follicular lymphoma and diffuse large B-cell lymphoma
Authors: Fang Douglas D. , Zhai Guoqin , Gu Shoulai , Tao Ran , Tang Qiuqiong , Min Ping , Wang Qixin , Yang Dongmei , Gu Jiaxing , Li Yinfeng , Chen Dingxiong , Li Jiajun , Wang Guangfeng , Yang Dajun , Zhai Yifan
Published: August 2019

Follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) are among the most prevalent B-lymphocyte neoplasms with Bruton’s tyrosine kinase (BTK) often found abnormally activated in these patients. BTK inhibitor ibrutinib has showed proven efficacy in both indications, but its clinical application is limited to gradually acquired resistance. Recent studies reported that ibrutinib-resistant cells exhibited higher BCL-2 expression and therefore increase their sensitivity to BCL-2 inhibitors. In this study, using a BCL-2 selective inhibitor APG-2575, we asked whether its combination with ibrutinib enhanced antitumor activity in FL and DLBCL in preclinical studies. Indeed, the combination treatment with APG-2575 and ibrutinib synergistically enhanced antitumor activity in DOHH2 cell-derived FL xenograft models. Similarly, enhanced activity was achieved in DLBCL xenograft models derived from OCI-LY1 cells. Interestingly, in OCI-LY1 xenografts, the combination treatment achieved a 100 % response rate, which include partially (PR) and complete tumor regression (CR), with ~80 % of CR and ~20 % of PR recorded. Durable response was continuously observed till the end of the study after dose suspension. Pharmacokinetic studies revealed that there were no significant changes in drug concentrations between single agents and the combination treatment, suggesting there was no drug-drug interaction between these two agents. On-target pharmacodynamic (PD) modulations of BTK and apoptosis pathways were observed in corresponding tumor tissues co-treated with ibrutinib and APG-2575. An increase in the levels of cleaved PARP-1 protein was observed under the combination treatment suggesting enhanced induction of apoptosis occurred, which may be the biological event contributing to the synergistic effect of the combination treatment. In summary, our study suggests that APG-2575 may be applied to the combination therapy with ibrutinib in the treatment of FL and DLBCL.

Citation Format: Douglas D. Fang, Guoqin Zhai, Shoulai Gu, Ran Tao, Qiuqiong Tang, Ping Min, Qixin Wang, Dongmei Yang, Jiaxing Gu, Yinfeng Li, Dingxiong Chen, Jiajun Li, Guangfeng Wang, Dajun Yang, Yifan Zhai. BCL-2 selective inhibitor APG-2575 synergizes with BTK inhibitor in preclinical xenograft models of follicular lymphoma and diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2058.
Abstract 2074: Bioactive marine drugs target acquired oncogenic burden in resilient pancreatic cancer
Authors: Aravindan Sheeja , Somasundaram Somasundaram T. , Natarajan Mohan , Herman Terence S. , Aravindan Natarajan
Published: August 2019

The upsurge of marine-derived therapeutics for cancer treatment is evident, with many drugs in clinical use and in clinical trials. Seaweeds harbor large amounts of polyphenols and their anti-cancer benefit is linear to their anti-oxidant activity. Our studies identified three superlative anti-cancer seaweed polyphenol drug candidates (SW-PD). We investigated the acquisition of oncogenic burden in radiotherapy-resilient pancreatic cancer (PC) that could drive tumor relapse, and elucidated the efficacy of SW-PD candidates as adjuvants in genetically diverse in vitro systems (Panc-1, Panc-3.27, BxPC-3, MiaPaCa-2) and a mouse model of radiation-residual disease. For residual PC studies, PC xenografts developed in nude mice were selectively exposed to clinically relevant fractionated irradiation, (2 Gy/day for 5 days per week for a total of 3 weeks), to a total dose of 30 Gy. QPCR profiling of custom archived 88 oncogenes in therapy-resilient PC cells identified a ‘shared’ activation of 40 oncogenes. SW-PD pre-treatment inflicted a significant mitigation of acquired (shared) oncogenic burden, in addition to drug- and cell-line-specific repression signatures. Tissue-microarray with IHC of radiation-residual tumors in mice signified acquired cellular localization of key oncoproteins (Fosb, cFos, Fra2, cMYC, MYB, NF2) and other critical architects (SELE, CD73, Cytokeratins, Col3, Decorin, and Sulf2). Conversely, SW-PD treatment inhibited the acquisition of these critical drivers of tumor genesis, dissemination, and evolution. Heightened death of resilient PC cells with SW-PD treatment validated the translation aspects. The results defined the acquisition of oncogenic burden in resilient PC and demonstrated that the marine polyphenols effectively target the acquired oncogenic burden and could serve as adjuvant(s) for PC treatment.

Citation Format: Sheeja Aravindan, Somasundaram T. Somasundaram, Mohan Natarajan, Terence S. Herman, Natarajan Aravindan. Bioactive marine drugs target acquired oncogenic burden in resilient pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2074.
Abstract 2076: Targeting vulnerable aberrant cancer-associated pathways by rationalized triple combination regimens to effectively control urothelial carcinoma cells in vitro and in vivo
Authors: Wang Hwa-Chain R. , Pattarawat Pawat , Wang Jinquan , Donnell Robert
Published: August 2019

Urinary bladder cancer (UBC) is the fifth-most common cancer in the United States, and urothelial carcinomas (UCs) account for more than 90% of UBCs. Although current therapies are effective in the short-term treatment of UCs, the high tendency (>50%) for them to recur and progress to life threatening, advanced muscle-invasive UCs requires life-long surveillance with extended medical care. Thus, it still requires extensive therapeutic development to achieve an effective control of UC recurrence and improve the response rate and overall survival for UC patients. To develop effective and safe regimens to control the development and recurrence of advanced UCs, we investigated the efficacy of rationalized combination regimens in controlling UC cells in vitro and UC cell-derived xenografts (CDXs) in vivo. Our in vitro studies identified a triple combination of gemcitabine, romidepsin, and cisplatin, which are all FDA-approved anticancer agents, effective in synergistically inducing death and reducing drug resistance of various UC cell lines. Our studies also revealed that UC-associated aberrant pathways, including mitochondrial reactive oxygen species (ROS) and the ERK-Nox pathway, which were already induced in UC cells, were enhanced by the triple combination regimen to play essential roles for inducing apoptosis and reducing resistance. We also detected that the unfolded protein response played a role in modulating cell death induced by the regimen. Our in vivo studies used a tolerable protocol for administering combination regimens into animals. Our studies verified the efficacy of the triple combination gemcitabine plus romidepsin and cisplatin regimen, versus double combination regimens, in controlling UC CDXs in immune-deficient mice. In conclusion, our studies indicated that a rationalized triple combination of gemcitabine plus romidepsin and cisplatin regimen was more effective than double combination regimens in modulating UC-associated vulnerable aberrant pathways and controlling UC tumor development and recurrence in animals with low tolerable/ reversible adverse effects. Thus, the triple combination gemcitabine plus romidepsin and cisplatin regimen should be promptly considered as an advanced treatment over the conventional double combination gemcitabine plus cisplatin regimen in clinical trials in order to control advanced UCs.

Citation Format: Hwa-Chain R. Wang, Pawat Pattarawat, Jinquan Wang, Robert Donnell. Targeting vulnerable aberrant cancer-associated pathways by rationalized triple combination regimens to effectively control urothelial carcinoma cells in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2076.