bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5032: Attenuated Salmonella blocks cancer metastasis by activating NK cells in an IFN-γ-dependent manner
Authors: Lin Qiubin , Rong Li , Huang Jiandong
Published: September 2020

Introduction and objectives:

The recent remarkable clinical results of cancer immunotherapies, such as immune checkpoint blockade and chimeric antigen receptor T cells (CAR-T), has sparked a great deal of enthusiasm in manipulating the immune response in the fight against cancer. Bacterial cancer therapies represent an alternative approach to targeting solid tumours for evoking anti-tumour immune responses. As a facultative anaerobic pathogen that can penetrate and colonize solid tumours, Salmonella typhimurium gained lot of interest in the investigation of bacterial cancer therapies and can exert its intrinsic antitumor activity by directly inducing systemic immune responses. However, most bacterial cancer therapies, including salmonella-based cancer therapies, have mainly focused on the suppression of already established tumours rather than metastasis.

Metastasis accounts for 90% of cancer related deaths and blocking of metastatic cascade has critical clinical impact. However, the clinical drug development for cancer treatment, including cancer immunotherapies, is evaluated largely depending on their ability to cause tumour shrinkage and ignores the effect on metastasis as it has proven challenging to target. Therefore, there is an urgent need for novel therapeutic strategies and agents targeting metastasis.

Experimental procedures and outcomes

Using an attenuated Salmonella typhimurium strain YB1 engineered by our lab, we have found a potent suppressive effect of attenuated salmonella on cancer metastasis, regardless of cancer types and genetic background, by evoking strong anti-metastatic immune response. Further study showed that suppression of cancer metastasis by attenuated Salmonella only requires the innate immune response. Among the many induced cytokines, we identified IFN-γ as an indispensable factor for inhibiting cancer metastasis. Based on CyTOF (mass cytometry or cytometry by time of flight) analysis of the innate immune responses after Salmonella treatment and antibody-mediated cell depletion, we further demonstrated that NK cells are the major factor involved in Salmonella-provoked metastasis suppression. We found that IFN-γ was mainly produced by NK cells during early Salmonella infection, and in turn, IFN-γ promoted the accumulation, activation and cytotoxicity of NK cells. The IFN-γ-dependent NK cells directly eliminated newly accumulated cancer cells in the lung to block the cancer metastasis cascade in response to the Salmonella treatment.

Citation Format: Qiubin Lin, Li Rong, Jiandong Huang. Attenuated Salmonella blocks cancer metastasis by activating NK cells in an IFN-γ-dependent manner [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5032.
Abstract 5039: SCD1/FADS2 and ferroptosis signalings orchestrate ovarian cancer cell metabolism and metastatic progression in peritoneal metastases
Authors: Xuan Yang , Yung Mingo MH , Chen Arvin FS , Ngan Hextan YS , Chan David W
Published: September 2020

Peritoneal metastases are common and usually associated with poor prognosis in advanced ovarian cancers. Emerging evidence indicates that the ascites microenvironment provides a key reservoir of growth factors and free fatty acids for promoting metastatic recurrence and chemoresistance of ovarian cancers. However, how the metastatic ovarian cancer cells adapt and utilize such a unique tumor microenvironment remains elusive. Here, we report that ovarian cancer cells co-cultured in ascites or omental conditioned medium (OCM) enhance their oncogenic capacities through elevated lipid metabolism. RNA sequencing (RNA-seq) and lipidomic profiling analyses revealed two key mediators of UFAs, Stearoyl-CoA Desaturase-1 (SCD1) and Fatty acid Desaturase-2 (FADS2) were upregulated accompanied with the accumulation of poly- and mono-unsaturated fatty acids (UFAs) in ovarian cancer cells co-cultured in OCM. Enforced expression of either SCD1 or FADS2 promotes cancer stemness, in vitro and ex vivo tumor colony formation, and cell migration/invasion through activation of NFκB signaling. SCD1/FADS2 increases not only UFAs accumulation but also the cell membrane fluidity which is crucial for the epithelial-mesenchymal transition (EMT) and metastatic potentials. On the other hand, RNA-Seq analysis on SCD1/FADS2-depleted ovarian cancer cells showed the ferroptotic activities were inversely correlated with SCD1/FADS2. Of note, either knockout of SCD1/FADS2 or co-treatment of Erastin could enhance cisplatin-induced cytotoxicity through the elevation of iron-mediated reactive oxygen species (ROS), and subsequently led ferroptosis by upregulation of GPX4/xCT expressions in ovarian cancer cells. Higher tumorigenic capacity expressed in iron-rich ovarian cancer cells, which contains sphere formation, EMT potential and cell viability. Taken together, SCD1/FADS2 and ferroptosis signaling pathways orchestrate the metabolic activities, ROS stresses and cellular iron homeostasis during the metastatic progression of ovarian cancer cells in peritoneum microenvironment. Hence, targeting these signaling cascades by the drug cocktail/combination strategy could reduce the risk of metastatic recurrence and chemoresistance in advanced ovarian cancers.

Citation Format: Yang Xuan, Mingo MH Yung, Arvin FS Chen, Hextan YS Ngan, David W Chan. SCD1/FADS2 and ferroptosis signalings orchestrate ovarian cancer cell metabolism and metastatic progression in peritoneal metastases [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5039.
Abstract 504: p27 transcriptionally coregulates STAT3 to drive cancer stem cells
Authors: Razavipour Seyedehfatemeh , Jang Kibeom , Yoon Hyunho , Kim Minsoon , Shin Miyoung , Zhao Dekuang , Slingerland Joyce
Published: September 2020

p27 is a cell cycle inhibitor and a tumor suppressor. It can also regulate cellular processes including migration and transcription through mechanisms independent of its CDK-inhibitory role. In cancers, p27 C-terminal phosphorylation by PI3K-activated kinases, AKT, RSK1, and SGK1 at T157 and T198 alters p27 protein-protein interactions and shifts p27 from CDK-inhibitor to an oncogene. Previously, our group showed that CDK-binding defective p27pT157pT198 phosphomimetic (p27CK-DD) upregulates epithelial-mesenchymal transition (EMT) and the metastatic potential of cancer cell lines. In addition to its action to promote EMT, p27 appears to promote CSC expansion and or maintenance. Here we demonstrated that C-terminally phosphorylated p27 increases CSC properties, including tumor sphere formation and CSC markers. p27CK-DD increases the expression of several embryonic stem cell transcription factors (ES-TFs), including SOX2, NANOG and cMYC, which are known to drive embryonic stem cell self-renewal and to promote CSC expansion. A human phospho-kinase array showed Pyk2 is activated by p27CK-DD. We demonstrated that Pyk2 activation and its binding to p27 are dependent on phosphorylation of p27 at T198 and T157. Treatment with a Pyk2 inhibitor, and PYK2-knockdown by siRNA or shRNA revealed that Pyk2 is a key mediator of the increase in tumor spheres, ALDH1 activity and ES-TFs in cancer cells expressing abundant C-terminally phosphorylated p27. Pyk2 enhances STAT3 phosphorylation and activation. We identified that C-terminally phosphorylated p27 recruits STAT3 to form p27-Pyk2-STAT3 complex leading to STAT3 activation. p27/STAT3 complexes co-localize to the nucleus and co-occupy chromatin. We confirmed by ChIP-qPCR that p27/STAT3 bind and activate the cMYC promotor. Altogether, in breast cancer cells with high p27pT157pT198 or expressing p27CK−DD, STAT3 is partly activated by Pyk2 and interacts with p27. p27 is a STAT3 coregulator, whose assembly and chromatin association is governed by p27 phosphorylation. These data reveal a novel mechanism whereby p27-driven Pyk2 activation promotes CSC expansion and tumor progression via transcriptionally activation of the STAT3 and its target genes.

Citation Format: Seyedehfatemeh Razavipour, Kibeom Jang, Hyunho Yoon, Minsoon Kim, Miyoung Shin, Dekuang Zhao, Joyce Slingerland. p27 transcriptionally coregulates STAT3 to drive cancer stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 504.
Abstract 5045: Predictive factors for successful growth of patient derived xenograft (PDX)
Authors: Hernandez Tatiana , Baños Natalia , Bonilla Victoria , del Puerto Laura , Doger Bernard , Garcia-Foncillas Jesus , Wick Michael , Moreno Victor
Published: September 2020

Background: PDXs have become a core component of translational cancer research. Developing these models can become challenging since little is known about which factors influence engraftment rates. We sought to determine which clinical, pathological or molecular factors may predict a higher chance of success for PDX development.

Methods: Between March 2017 and August 2019, biopsies obtained from patients with primary or metastatic cancer were implanted into athymic nude mice. Statistical analyses were performed to identify factors that could correlate with final engraftment defined as tumor volume of 150 mm3 in at least three consecutive measurements. We focus on clinical (patient factors) pathological (tumor sample) and molecular characteristics (tumor sample). Information regarding sampling process was also analyzed.

Results: 440 tumor samples were collected and implanted. 15 failed due lack of tumor cells. Of 425 tumor-positive samples, 143 PDXs achieved successful growth at time of analysis. The following clinical characteristics were correlated with engraftment success: systemic steroid administration within 2 weeks of sampling (45.5% (30/66) within the steroids-receiving group achieved growth vs 31% (113/359) no-steroids) (p: 0.027). High neutrophils/lymphocytes (NLR) ratio (>5) (42.3% (47/111) of tumors with NLR>5 achieved growth vs 30% (96/313) in the NLR<5 group; P:0,018). Regarding pathological tumor characteristics, higher ki67 levels were correlated with better engraftment rates (Low (Ki67<15%): 10% (5/46) vs. Medium (Ki67:15-30%): 25% (7/28) and high (Ki67>30%): 34% (16/47) (p:0.029). The presence of mucinous cells (signet ring cells) was also correlated with higher chance of tumor engraftment (50% (19) vs. 32% (124/387) (p: 0.022). Negativity for estrogen receptors could also be a positive predictive marker (14% (8/57) of ER+ achieved growth vs 46.7% (7/15) ER negative (p: 0.011). Lastly, sampling source (either primary of metastatic tumors) was not correlated with successful growth.

Conclusions: tumors with higher Ki67, mucinous features and ER expression negative have higher chance of PDX development. Some clinical characteristics can also interfere with PDXs development such as use of steroids or NLR.

Citation Format: Tatiana Hernandez, Natalia Baños, Victoria Bonilla, Laura del Puerto, Bernard Doger, Jesus Garcia-Foncillas, Michael Wick, Victor Moreno. Predictive factors for successful growth of patient derived xenograft (PDX) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5045.
Abstract 5066: Investigating the role of IL-6 to modulate the immunosuppressive tumor microenvironment of EGFR mutant NSCLC
Authors: Patel Sonia A. , Nilsson Monique , Cho Sungnam , Le Xiuning , Heymach John
Published: September 2020

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death with a less than 15% five-year survival. Approximately 15% of NSCLC cases in the US harbor epidermal growth factor receptor (EGFR) activating mutations and are initially highly sensitive to EGFR tyrosine kinase inhibitors (TKI). Unfortunately, the vast majority of tumors acquire EGFR TKI resistance, and treating resistant disease remains a clinical challenge. Additional therapeutic modalities, such as immunotherapy, are needed to improve clinical outcome. While immunotherapy has shown clinical activity in multiple cancer types including NSCLC, a lack of efficacy has been observed in EGFR mutant NSCLC patients. The biological mechanisms driving immunotherapy resistance in EGFR mutant NSCLC are not well understood, and additional EGFR mutant NSCLC patients. We previously reported that IL-6 is upregulated in NSCLC cells with acquired resistance to EGFR TKIs. Given the role of IL-6 as an immunomodulatory cytokine, we evaluated the effects of IL-6 signaling on immunosuppression in genetically engineered mouse models (GEMMs) of EGFR mutant NSCLC. IL-6 knockout (EGFRL858R/IL-6KO) resulted in decreased PD-L1 expression on the surface of EGFR mutant tumor cells compared to tumors in IL-6 proficient mice. Additionally, we observed a modest increase in PD-1 expression on CD8+ T cells in EGFRL858R/IL-6KO mice compared to EGFRL858R mice, suggesting that IL-6 acts on both tumor and immune populations to facilitate a “cold” immunosuppressive tumor microenvironment. We next analyzed immune populations in EGFR mutant tumors from mice with or without knockout of IL-6 by single cell RNA sequencing and validated these findings by immunohistochemistry. Tumors in EGFRL858R/IL-6KO mice displayed decreased B-cell (CD20) populations, increased macrophages (F480), and increased natural killer cells (NKp46) compared to tumors from IL-6 expressing EGFRL858R mice. Our data suggests that the increased expression of IL-6 in EGFR TKI resistant NSCLC may alter immune cell populations within the tumor microenvironment and impact immunosuppression, and that therapeutic approach targeting IL-6 merit further investigation for EGFR-mutant NSCLC.

Citation Format: Sonia A. Patel, Monique Nilsson, Sungnam Cho, Xiuning Le, John Heymach. Investigating the role of IL-6 to modulate the immunosuppressive tumor microenvironment of EGFR mutant NSCLC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5066.
Abstract 5086: The aged tumor microenvironment promotes melanoma metabolic plasticity and therapy
Authors: Alicea Gretchen Marie , Rebecca Vito W. , Goldman Aaron , Fane Mitchell , Behera Reeti , Webster Marie , Ecker Brett , Blair Ian , Speicher David , Schug Zachary , Weeraratna Ashani
Published: September 2020

“Aged” melanoma patients (>55 years old) have poorer prognosis and reduced response rates to targeted therapy relative to “young” patients (<40 years old). Here, we report an altered metabolic landscape in the aged tumor microenvironment (TME) as critical for melanoma aggressiveness. Aged fibroblasts uptake high levels of glucose compared to their young counterparts, which fuels lipid production. Melanoma cells cultured in an aged TME in vitro or in vivo display elevated intracellular lipid levels and increased metastatic potential relative to melanoma cells in a young TME. Further, lipidomics reveal an elevated lipid profile (i.e., triglycerides, ceramides, and cholesterol) preferentially secreted by aged fibroblasts relative to young. In turn, we show that melanoma cells adaptively increase extracellular fatty acid scavenging via the lipid transporter FATP2 in the lipid rich aged TME. Melanoma cells cultured in the aged TME also display an increased oxygen consumption rate (OCR) relative to those grown in young TME, which could be ablated when fatty acid transport into the mitochondria is blocked with etomixir. Notably, melanoma cells in the aged TME treated with BRAFi/MEKi in combination with a FATP2 inhibitor display decreased OCR relative to BRAFi/MEKi inhibitor alone, which paralleled the increased overall survival of aged mice treated with BRAFi/MEKi/FATP2i relative to either agent alone. To rule out off-target effects of the FATP2 inhibitor, we used a doxycycline inducible system for FATP2 knockdown in melanoma cells and reproduced the abrogation of tumor growth and extension of survival in aged immune-competent mice when combined with BRAFi/MEK therapy. Additionally, elevated FATP2 levels correlate with worse response in human patients. We hypothesize the aged TME triggers adaptive metabolic plasticity of melanoma cells critical for therapy escape.

Citation Format: Gretchen Marie Alicea, Vito W. Rebecca, Aaron Goldman, Mitchell Fane, Reeti Behera, Marie Webster, Brett Ecker, Ian Blair, David Speicher, Zachary Schug, Ashani Weeraratna. The aged tumor microenvironment promotes melanoma metabolic plasticity and therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5086.
Abstract 509: Discovery of novel small molecule inhibitors of Wnt signaling through in silico molecular docking
Authors: Low Joo-Leng , Du Weina , Gocha Tenzin , Gokce Oguz , Zhang Xiaoqian , Yim Daniel G. , Ramasamy Adaikalavan , Fan Hao , DasGupta Ramanuj
Published: September 2020

Canonical Wnt signaling is one of the crucial pathways involved in embryonic development and tissue homeostasis. However this pathway is often dysregulated in cancers and thus developing effective inhibitors of Wnt signaling is of great interest in the field. Our strategy for inhibition of Wnt signaling involves disruption of the β-catenin (β-cat)-Tcf4 protein-protein interaction (PPI) complex, which is the key effector for nuclear Wnt signaling activity. Towards this goal, we developed a computational model of β-cat for predicting small molecule binders. Application of our computational model to an in silico screen against a library of more than 10,000 compounds predicted 27 compounds as good binders to β-cat. Through in vitro validation experiments such as Wnt reporter assays, co-immunoprecipitation, and surface plasmon resonance (SPR) studies, we identified compound GB1874 as our most promising candidate for inhibiting the β-cat-Tcf4 PPI. Compound GB1874 also affected the proliferation of Wnt-addicted colorectal cancer (CRC) cells in vitro and in tumor xenograft models in vivo. Notably the in vivo efficacy was associated with minimal systemic toxicity. In summary, we report the identification of a novel disruptor of β-cat-Tcf4 interaction, GB1874, which can serve as a promising small molecule candidate for a Wnt signaling inhibitor. Moreover, our study demonstrates the feasibility of using computational modeling to identify new inhibitors of PPI.

Citation Format: Joo-Leng Low, Weina Du, Tenzin Gocha, Oguz Gokce, Xiaoqian Zhang, Daniel G. Yim, Adaikalavan Ramasamy, Hao Fan, Ramanuj DasGupta. Discovery of novel small molecule inhibitors of Wnt signaling through in silico molecular docking [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 509.
Abstract 5091: TIMP-1 in tumor-associated fibroblasts drives tumor progression in lung adenocarcinoma through CD63 interaction
Authors: Duch Paula , Gabasa Marta , Ikemori Rafael , Arshakyan Marselina , Hillberg Frank , Reguart Noemí , Radisky Derek , Alcaraz Jordi
Published: September 2020

Tumor associated fibroblasts (TAFs) are important regulators of tumor growth and resistance to therapies. We have recently shown that lung TAFs in vitro respond to the antifibrotic drug nintedanib in adenocarcinoma (ADC) but not squamous cell carcinoma (SCC). We also showed that the tumor-promoting effects of TAFs are driven by different mechanisms in ADC and SCC, which remain to be elucidated. Tissue inhibitor of metalloproteinases 1 (TIMP-1) has been associated with poor prognosis in lung cancer, its expression is downregulated by nintedanib, and our preliminary results reveal that its putative receptor, CD63, is overexpressed in ADC patients compared to SCC, supporting a selective crosstalk between TAFs and cancer cells in ADC through TIMP-1 and CD63. The aim of this study was to test this hypothesis using in vitro preclinical models. Primary fibroblasts were obtained with the patient informed consent, and using protocols approved by the Ethics Committee of the Hospital Clinic. ADC-TAFs and SCC-TAFs were stimulated with TGF-β1 in the presence or absence of nintedanib, and the TIMP-1 content in their conditioned medium was determined by ELISA. TIMP-1 was knocked-down in ADC-TAFs by siRNA, and the corresponding conditioned medium was used to stimulate growth and invasion of the high-CD63 ADC cell line, H1437. Likewise, CD63 expression in H1437 cells was reduced by siRNA. Our in vitro results showed that TIMP-1 secretion induced by TGF-β1 is significantly larger in ADC-TAFs compared to SCC-TAFs. Likewise, nintedanib elicited a higher downregulation of secreted TIMP-1 in ADC-TAFs compared to SCC-TAFs. Of note, TIMP-1 from ADC-TAFs was necessary to induce growth and invasion of H1437 cells. Likewise, knocking-down CD63 in H1437 ADC cells was sufficient to reduce the growth and invasion elicited by the conditioned medium of TGF-β1 activated ADC-TAFs. Collectively, our results unveil a novel stroma-carcinoma crosstalk driven by TIMP-1 and CD63 selectively in lung ADC, and support that such heterotypic crosstalk may underlie the aberrant tumor-promoting effects of ADC-TAFs that are selectively downregulated by nintedanib.

Citation Format: Paula Duch, Marta Gabasa, Rafael Ikemori, Marselina Arshakyan, Frank Hillberg, Noemí Reguart, Derek Radisky, Jordi Alcaraz. TIMP-1 in tumor-associated fibroblasts drives tumor progression in lung adenocarcinoma through CD63 interaction [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5091.
Abstract 5115: Cancer-associated fibroblasts under the influence of melanoma cells might stimulate the lymphatic vessel formation
Authors: Maeda Shusaku , Yashiro Masakazu , Motomura Hisashi , Hatano Takaharu , Fujikawa Heishiro
Published: September 2020

Introduction: Cancer-associated fibroblasts (CAFs) in tumor microenvironment have an interaction with tumor cells and play an important role for the progression and metastasis. Tumor lymphangiogenesis may be controlled by not only tumor cells but also tumor microenvironment, and some reports suggested that CAFs may promote tumor lymphangiogenesis. In malignant melanoma, lymph node metastasis is important prognostic factor. However, no study about the interaction among CAFs, tumor cells, and human lymphatic endothelial cells (HLECs) was found. In this study, we evaluated the effect of CAFs on lymphatic vessel formation in malignant melanoma. Materials and methods: We used CAFs established from a primary malignant melanoma, HLECs, and two melanoma cell lines (MMAc, COLO679). CAFs and melanoma cell lines were cultured with DMEM. Serum-free conditioned medium (CM) was obtained from CAFs (CAF-CM) and two melanoma cells (MMAc-CM, COLO679-CM). Also, CM from CAFs cultured with MMAc-CM (MMAcCAF-CM) and COLO679-CM (COLO679CAF-CM) was obtained. HLECs were cultured by endothelial growth medium (EGM) in combination with 50% of each CM. Control group was cultured with 50% EGM and 50% of serum-free endothelial basal medium. The effect of each CM on the proliferation of HLECs was examined by CCK assay and tube formation assay. Tube formation was assessed by counting total tube length, number of tubes and junctions at 12 hours of incubation. Result: Both MMAcCAF-CM and COLO679CAF-CM significantly increased the proliferation of HLECs in compared with the control or CAF-CM alone, after 24, 48 and 72 hours of incubation. In tube formation assay, both tube length and number of tubes formed by HLECs were significantly longer and larger in MMAcCAF-CM and MMAc-CM group than control group. The tube length, number of tubes and junctions treated by MMAcCAF-CM or COLO679CAF-CM were longer and larger than in control and CAF-CM group. Conclusion: CAFs under the influence of melanoma cells might have a more progressive effect on HLECs, resulting in promotion of lymphangiogenesis.

Citation Format: Shusaku Maeda, Masakazu Yashiro, Hisashi Motomura, Takaharu Hatano, Heishiro Fujikawa. Cancer-associated fibroblasts under the influence of melanoma cells might stimulate the lymphatic vessel formation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5115.
Abstract 5131: Differentially expressed proteins in esophageal squamous cell carcinoma tissue
Authors: Si Fuchun
Published: September 2020

Purpose: To identify differentially expressed proteins among the esophageal squamous cell carcinoma(ESCC) carcinoma tissue, adjacent normal tissue, normal esophageal mucosa, in order to look out the protein related to different stages and study the molecular mechanism and occurrence of the esophageal squamous cell carcinoma.

Methods: Tissue specimens of ESCC and the corresponding clinical data database of ESCC patients were established, a total of 69 patients were selected according to distribution of their sex and age. Two-dimensional gel electrophoresis technology to electrophoresis and get the separation protein of cancerous tissues, adjacent tissues and normal esophageal tissues,then ImageMaster 2D Platinum software was used to search, quantify, match and statistical analysise the proteins on the gel image. 4700 Proteomics Analyzer (TOF/TOFTM) was used to analyze protein spots, in which differentially expressed proteins meet standards were further searched in the Matrix Science, London, UK protein database.

Results: Totle 44 and 29 kinds of differentially expressed proteins were screened from esophageal cancer tissues, adjacent noncancerous tissues and normal tissues, among which, there were 13 differentially expressed proteins between esophageal cancer tissues and adjacent noncancerous tissues, 14 differentially expressed proteins between esophageal cancer tissues and normal tissues, 17 between esophageal adjacent noncancerous tissues and normal tissues. These differential expression proteins mainly involved in PKC-catalyzed phosphorylation of inhibitory phosphoprotein of myosin phosphatase, nuclear factor of activated T cells(NFAT) pathway, Integrin Signaling Pathway, Cell to Cell Adhesion Signaling, Calmodulin(CaM) nuclear factor of activated T cells, Mineral absorption of calcium binding protein path way(S100), Path way of vascular smooth muscle contraction affe cted by caldesmon, Apoptotic Signaling in Response to DNA Damage path way, which had the function of cytoskeleton structure, protein phosphorylation, acetylation of aminoend, regulation of calcium combination,tumor cell movement, signal transduction, cell differentiation, cell apoptosis, muscle contraction, translation regulation, gene expression, energy metabolism, cell proliferation.

Conclusion: This study study achieved differentially expressed protein in ESCC tissue patients, provides new basis for the study of esophageal cancer carcinogenesis mechanism.

Citation Format: Fuchun Si. Differentially expressed proteins in esophageal squamous cell carcinoma tissue [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5131.
Abstract 5140: The expression and function of LAP2 and its interaction with Chmp1A in pancreatic cancer
Authors: Park Maiyon
Published: September 2020

Introduction: Chromatin modifying protein 1A (Chmp1A) suppresses pancreatic cancer growth by the activation of ATM and p53. To understand the mechanisms underlying the function of Chmp1A in pancreatic cancer, Chmp1A associated proteins were identified using proteomic analysis. Lamina-associated polypeptide 2 (LAP2) protein isoforms were found to interact with Chmp1A. LAP2 has been shown to interact with Retinoblastoma (Rb) tumor suppressor and shown to be up-regulated in most proliferating cancer cells. However, the function of LAP2 in pancreatic cancer has been poorly understood. In this study, we propose that LAP2 protein functions as tumor suppressor by interacting with Chmp1A in pancreatic cancer.

Experimental procedures: LAP2 transcripts were determined by RT-PCR and LAP2 proteins by WB and microscopic analysis in pancreatic cancer cells compared to normal pancreatic cells. We then tested the function of LAP2 by examining the effect of its overexpression on cancer cell growth. We also tested the interaction between Chmp1A and LAP2 by examining LAP2 expression in Chmp1A overexpressing or silenced cells and vice versa.

Results: The level of LAP2 transcripts and proteins was increased in pancreatic cancer, compared to normal cells. Interestingly, LAP2 protein level was increased in cells derived from well, but not poorly differentiated pancreatic cancers. Immunostaining demonstrated an increased and altered cellular expression of LAP2, compared to normal. LAP2 expression was increased in Chmp1A overexpressing cells but decreased in Chmp1A silenced cells. In addition, Chmp1A expression was increased in LAP2 overexpressing cells.

Summary: The expression of LAP2 isoforms were increased in tumor cells, compared to normal pancreatic cells. LAP2 overexpression study suggests its functions as tumor suppressor in pancreatic cancer. Immunocytochemical analysis suggests that the increased and altered expression of LAP2 might be important for its function in pancreatic cancer. Our data also indicates that there is comparable change in the expression between Chmp1A and LAP2 in pancreatic cancer cells.

Citation Format: Maiyon Park. The expression and function of LAP2 and its interaction with Chmp1A in pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5140.
Abstract 5176: A novel, completely human fusion construct containing active Granzyme B efficiently kills Folate Receptor alpha positive tumor cells
Authors: de Cienfuegos Ana Alvarez , Cheung Laurence H. , Mohamedali Khalid A. , Hittelman Walter N. , Rosenblum Michael G.
Published: September 2020

Folate receptor alpha (FRα) is an attractive therapeutic target because it is highly overexpressed on several tumors such as ovarian, breast and lung cancers and demonstrates low or restricted distribution on normal tissues. In addition, FRα has been shown to play a tumor promoting role with its expression associated with poor prognosis of cancer patients. In our laboratory, we have generated two new molecules targeting FRα and containing active granzyme B (GrB) as the cytotoxic payload. We used as binding domains two different scFvs targeting the FRα from antibodies used in clinical trials. These human scFvs (designated mov003 and mov018) were fused to human GrB through an engineered IgG heavy-chain fragment (designated as Fc) which contains a dimerization domain. The constructs were cloned into the pSECTag vector containing an upstream 5 kDa purification tag with an enterokinase (EK) cleavage site for facile removal. The molecules were expressed in transiently-transfected HEK-293E suspension cells over 3 days at 37°C in 5% CO2. The molecules were isolated from culture media and purified by ion-exchange chromatography followed by EK cleavage to remove the free tag. The yields were 12-18 mg/L for both molecules. The assessment of the specific binding of each molecule to the antigen target is ongoing. The cytotoxicity of the constructs against a panel of cell lines expressing various levels of FRα showed a specific cytotoxic effect with an IC50 in the low nanomolar range (5-100 nM), particularly exhibiting very low IC50 values against breast (MDA-MB-231) and ovarian cell lines (IGROV-1). Receptor negative cells demonstrated IC50 levels in the micromolar range. Based on IC50 values, these data demonstrate suggest the fusion constructs targeting FRα are efficiently internalized and effectively deliver the active serine protease to the cytosol, activating the cytotoxic cascade. In vitro stability assays over 96 hours showed that both constructs are highly stable in mouse serum. Immunofluorescence and confocal studies to examine the time course of internalization and the pro-apoptotic events triggered by these agents are under examination and will be presented. Research conducted, in part, by the Clayton Foundation for Research.

Citation Format: Ana Alvarez de Cienfuegos, Laurence H. Cheung, Khalid A. Mohamedali, Walter N. Hittelman, Michael G. Rosenblum. A novel, completely human fusion construct containing active Granzyme B efficiently kills Folate Receptor alpha positive tumor cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5176.
Abstract 5246: Disulfiram anticancer metabolite, dithiocarb-copper complex, demonstrates anti-neoplastic activity against hypermutated colorectal cancers
Authors: Dumut Daciana Catalina , De Sanctis Juan B. , Mistrik Martin , Skrott Zdenek , Dzubak Petr , Bartek Jiri , Hajduch Marian , Radzioch Danuta
Published: September 2020

Introduction: Colorectal cancer (CRC) is the third leading cause of cancer related deaths in the United States. It was responsible for about 51,000 deaths during 2019. Surgical resection and systemic chemotherapy are standard approaches to treat both localized and metastatic CRC (mCRC). However, the effect of the chemotherapy depends on the characteristics of the individual tumor, for instance CRC subtypes with a microsatellite instability (MSI) tend to have better prognosis and response to both chemotherapy and immune check-point inhibition. Recently, Disulfiram's anti-cancer activity was explored and its metabolite ditiocarb-copper complex (CuET) was found to have high anti-cancer activity. The mechanism of action of CuET was proposed to act through p97 segregase adaptor NPL4 and activation of endoplasmic reticulum stress leading to preferential apoptosis of cancer cells. Since altered protein degradation pathway is targeted by CuET, this compound might display high efficacy in highly mutated colorectal cancers.

Methods: To assess CuET anti-cancer activity in vitro, we performed survival curve assays and colony-formation assays in both murine hypermutated colorectal cancer cell line MC-38 as well as in human KRAS mutant and MSI HCT116 cell line. We also evaluated anti-metastatic effect of CuET on both cell lines through migration-invasion assays in Boyden chamber setting. We assessed in vivo efficacy of systemic treatment with CuET alone or in combination with irinotecan, against MC-38 model in immunocompetent C57BL/6 mice. We characterized tumor growth, mice survival and tumor histopathology.

Results: Our results demonstrate that CuET displays cytotoxicity in MC-38 and HCT116 cells at very low doses with respective IC50 of 46nM and 88nM. CuET also significantly reduces the ability of both cell lines to grow and form colonies under sustained treatment. Additionally, CuET is effective in significantly reducing cancer cell migration and invasion through porous membrane in vitro. Furthermore, systemic treatment with CuET significantly reduced tumor growth in mice and prolonged survival when compared to controls.

Conclusion: CuET demonstrates cytotoxic activity against hypermutated colorectal cancer cells both in vitro and in vivo and should be further investigated in clinical trials.

Citation Format: Daciana Catalina Dumut, Juan B. De Sanctis, Martin Mistrik, Zdenek Skrott, Petr Dzubak, Jiri Bartek, Marian Hajduch, Danuta Radzioch. Disulfiram anticancer metabolite, dithiocarb-copper complex, demonstrates anti-neoplastic activity against hypermutated colorectal cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5246.
Abstract 5260: Preclinical studies with ONC201 in combination with clinically approved chemotherapy as a novel treatment strategy against Small Cell Lung Cancer (SCLC)
Authors: Lee Young S. , Zhou Lanlan , Azzoli Christopher , El-Deiry Wafik S.
Published: September 2020

According to the American Cancer Society, about 10-15 percent of all lung cancer diagnoses are small cell lung cancer (SCLC). The median survival rate of limited-stage small cell lung cancer is about 12 to 16 months. Several clinically approved chemotherapy drugs have been used to treat SCLC, with several more currently under clinical trials. However, a novel combination is still needed to increase patient life expectancy. ONC201 is an impridone, currently in clinical trials, that has proven to have proapoptotic effects in multiple tumor types. We hypothesized that treating SCLC cell lines with ONC201 in conjunction with clinically approved chemotherapy drugs, such as etoposide and carboplatin, will have a synergistic effect and serve as a potential novel combination treatment plan. SCLC cell lines H1048 and H1105 were treated for 72 hours with ONC201 and etoposide, or ONC201 and carboplatin. CellTiter-Glo cell viability assay and Western blotting of PARP cleavage and ATF4 were used to check synergy in the combination treatments. The combination of ONC201 and clinically approved chemotherapy drugs, etoposide and carboplatin, revealed that there was an upregulation in cleaved PARP and a synergistic effect on the overall cell samples. When looking at the combination index of the cell proliferation assays, the best index for each combination was 0.867 with an ONC201 dosage of 4 μM and an etoposide dosage of 0.05 μg/mL in H1048, 0.43191 at ONC201 dosage 0.3125 μM and etoposide dosage 2.5 μg/mL in H1105, and 0.20278 at ONC201 dosage 2.5 μM and carboplatin dosage 3.125 μg/mL in H1105. We are currently testing drug combinations including a triple drug combination of ONC201 and two clinically approved drugs and studying the mechanism underlying the synergies, with the potential to translate this novel combination treatment plan of ONC201 and clinically approved drugs into a clinical regimen to improve the outcome of SCLC patients.

Citation Format: Young S. Lee, Lanlan Zhou, Christopher Azzoli, Wafik S. El-Deiry. Preclinical studies with ONC201 in combination with clinically approved chemotherapy as a novel treatment strategy against Small Cell Lung Cancer (SCLC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5260.
Abstract 5294: Clinical utility of a multigene panel for hematologic malignancies
Authors: Bozdogan Sevcan Tug , Rencuzogullari Cagla , Mujde Cem , Bisgin Atil
Published: September 2020

Molecular profiling helps to diagnose, classify and guide the treatment of hematologic neoplasms. Thus NGS is the most powerful diagnostics tool to serve clinical management needs. In this study, we analyzed a multi-gene panel for genes associated with diagnostic, prognostic and therapeutic utilities for hematologic neoplasms. A total of 37 samples of myeloid and lenfoid were analyzed in GeneReader NGS system through a targeted multi-gene panel consisting of ASXL1, CALR, CBL, CEBPA, CSF3R, DNMT3A, EZH2, FLT3, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, RUNX1, SETBP1, SF3B1, SH2B3, SRSF2, TET2, TP53, U2AF1, ZRSR2. QCI-Analyze and QCI-Interpret were used for bioinformatic analyses. Each sample generated 2.176.058 million reads on average, and minimum of 99.5 % mapped reads to the reference reads. Total of 38 reportable variants of 16 genes were detected in 21 patients. The positivity rate was 70.5% (12 of 17) in myeloid group while its 90% (9 of 10 patients) in lymphoid group.In conclusion, our study shows the high sensitivity and specificity of the NGS technology that facilitates the identification of actionable genetic alterations on multiple genes simultaneously to avoid time-consuming multi-step procedures in hematologic malignancies.

Citation Format: Sevcan Tug Bozdogan, Cagla Rencuzogullari, Cem Mujde, Atil Bisgin. Clinical utility of a multigene panel for hematologic malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5294.
Abstract 5306: Clinicopathological significance of KIFC1 in human colorectal cancer
Authors: Akabane Shintaro , Oue Naohide , Fujiki Yuto , Uraoka Naohiro , Sakamoto Naoya , Sentani Kazuhiro , Egi Hiroyuki , Ohdan Hideki , Yasui Wataru
Published: September 2020

Colorectal cancer (CRC) ranks third in terms of incidence and second in terms of mortality, more than 1.8 million new colorectal cancer cases and 881,000 deaths are reported in Global Cancer Statistics 2018. Therefore, there is an urgent need for a new molecular target in considering treatment strategy. In recent years, cancer has been recognized as a stem cell disease. Spheroid colony formation has been demonstrated as an effective method to characterize cancer stem cells (CSCs). Previously, we analyzed the gene expression profile of spheroid colonies from the gastric cancer cell lines by microarray analysis and found that expression of Kinesin Family Member C1 (KIFC1) was upregulated in gastric spheroid colonies. The kinesin superfamily is a group of proteins that share a motor domain and participate in several biological functions. KIFC1 (or HSET) has been reported to play a vital role in centrosome clustering in cancer cells and is related to pathogenesis in several kinds of cancers. However, the expression of KIFC1 in CRC has not been investigated. The aim of this study is to investigate the expression and the biological function of KIFC1 in CRC. We analyzed the expression and distribution of KIFC1 in 129 CRC cases by immunohistochemistry. In contrast to weak or no staining of KIFC1 in non-neoplastic mucosa, CRC tissue showed stronger, more extensive KIFC1 staining. In total, 67 (52%) of 129 CRC cases were positive for KIFC1. KIFC1-positive CRC cases were significantly associated with CD44-positive and aldehyde dehydrogenase 1 (ALDH1)-positive CRC cases. In the CRC cell proliferation analysis, KIFC1 siRNA-transfected cells showed significantly reduced cell growth ability relative to negative control siRNA-transfected cells. The expression level of phosphorylated Akt and Erk were lower in KIFC1 siRNA-transfected cells compared to negative control siRNA-transfected cells. In the sphere formation analysis, the number and the size of spheres from CRC cells were significantly reduced in KIFC1 siRNA transfected cells than in negative control siRNA-transfected cells. These results indicate that KIFC1 plays an important role in CRC pathogenesis and might be involved in cancer stemness. Although further in-depth studies to clarify the biological significance of KIFC1 in colorectal CSCs are needed, KIFC1 may be a promising therapeutic target for the treatment of CRC.

Citation Format: Shintaro Akabane, Naohide Oue, Yuto Fujiki, Naohiro Uraoka, Naoya Sakamoto, Kazuhiro Sentani, Hiroyuki Egi, Hideki Ohdan, Wataru Yasui. Clinicopathological significance of KIFC1 in human colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5306.
Abstract 5308: Genetic characterization of sarcomatoid carcinomas reveals multiple novel actionable mutations and identifies KRAS mutation as a biomarker of poor prognosis
Authors: Ding Ying , Shao Yang W. , Na Chenglong , Yin Jiani C. , Hua Hongjin , Tao Ran , Jiang Yue , Hu Ran , He Xiao , Miao Chen , Zhu Dongqin , Zhang Zhihong
Published: September 2020

Background: Sarcomatoid component occurs in various epithelial malignancies and is associated with an aggressive disease course and poor clinical outcome. As it is largely rare, the molecular events underlying sarcomatoid carcinomas (SCs) remain poorly characterized. Here, we performed targeted next-generation sequencing (NGS) on patients with surgically resected SCs comprising distinct tissues of origin.

Methods: A total of 71 patients with pathological diagnosis of sarcomatoid carcinoma and underwent surgery were retrospectively enrolled in this study. Overall survival was defined as the time from surgery to death from any cause. Patients alive or lost to follow-up were censored. Genomic DNA from formalin-fixed paraffin-embedded samples was extracted for NGS and tumor mutation burden (TMB) analysis.

Results: In general, SCs occurred more commonly in males, except those of the gallbladder. SCs of the lung and the larynx were associated with a higher proportion of smokers (p=0.0015). Alterations in TP53, RB1, TERT and KRAS were highly frequent, with KRAS mutations being a biomarker of poor prognosis (median OS = 8 vs 16 months, p=0.03). Multiple novel alterations in potentially actionable genes, including ROS1 and NTRK1 fusions and ERBB2 amplification, were detected in the extra-pulmonary cohort. A relatively high proportion (30%) of extra-pulmonary SC patients had high TMB, with a median of 5.39 mutations per Mb. Lastly, copy number variations were common in SCs, and were non-overlapping between the primary and metastatic tumors.

Conclusions: Taken together, our results suggest that comprehensive genetic testing may be necessary to inform treatment options and identify prognostic biomarkers.

Citation Format: Ying Ding, Yang W. Shao, Chenglong Na, Jiani C. Yin, Hongjin Hua, Ran Tao, Yue Jiang, Ran Hu, Xiao He, Chen Miao, Dongqin Zhu, Zhihong Zhang. Genetic characterization of sarcomatoid carcinomas reveals multiple novel actionable mutations and identifies KRAS mutation as a biomarker of poor prognosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5308.