bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3100: Replacing fetal calf serum by human platelet lysate in cancer research and toxicology
Authors: Krämer Oliver H.
Published: August 2018

Experiments with cultured mammalian cells represent a common in vitro alternative to animal experiments. Fetal calf serum (FCS) is the most commonly used medium supplement. FCS contains a mixture of largely undefined growth factors and cytokines. Since FCS is received from unborn calves older than three months, it represents a massive burden for the pregnant cows and their fetuses. A needle is inserted into the heart of the fetus to collect blood. Since the animal is not under anesthesia, it may suffer pain and discomfort. Furthermore, the undefined nature of FCS is a source of experimental variation, undesired immune responses, and possible contaminations. Thus, alternative, defined, valid, and reliable medium supplements should be identified. We follow the principles of the 3Rs (Replacement, Reduction, Refinement). We established a method to receive human platelet lysate (hPL) from human blood platelets. We investigated if hPL represents potential true alternative for FCS in cell culture experiments and thereby the cruel slaughtering of unborn fetuses. hPL is received by freeze and thawing cycles and centrifugation steps. We successfully adapted three human leukemia cell lines (K562, NB4 and MV4-11) and HCT116 colon cancer cells to culture medium containing 1-5% hPL. We analyzed cell morphology by microscopy. Cell growth kinetics were examined by counting and cell cycle distributions were investigated by flow cytometry. Protein expression patterns were determined globally by global scale proteomics. We additionally determined possible FCS- or hPL-dependent reactions of cells to the well-established anti-cancer drugs Irinotecan, Hydroxyurea and Imatinib, and to epigenetic modulators of the HDAC inhibitor family (FK228 and the new, highly selective HDAC6 inhibitor Marbostat-100) by flow cytometry and Western blot. In these tests, we particularly focused on the expression of the pan-leukemic marker and transcription factor Wilms tumor 1 (WT1) and on the tumor suppressor p53. All tested cell lines grow with 5% FCS and 5% hPL. Cell morphology, cell cycle distribution, and protein expression patterns were not affected by such different culture conditions in resting and treated cells. We further show that the class I HDACs HDAC1-3, but not HDAC6, regulate WT1 and p53. Our findings demonstrate that hPL is a promising replacement for animal serum.

Citation Format: Oliver H. Krämer. Replacing fetal calf serum by human platelet lysate in cancer research and toxicology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3100.
Abstract 3104: Establishment of an oral tongue squamous cell carcinoma cell line from a never-smoking patient
Authors: Wang Steven J. , Asthana Saurabh , Zante Annemieke van , Olshen Adam B. , McCormick Frank , Tetsu Osamu
Published: August 2018

(Objective) The rising incidence of oral tongue squamous cell carcinoma (OTSCC) in patients who have never smoked and the paucity of knowledge of its biological behavior prompted us to develop a new cell line originating from a never-smoker. (Materials and methods) Fresh tumor tissue of keratinizing OTSCC was collected from a 44-year-old woman who had never smoked. Serum-free media with a low calcium concentration were used in cell culture, and a multifaceted approach was taken to verify and characterize the cell line, designated UCSF-OT-1109.(Results) UCSF-OT-1109 was authenticated by STR DNA fingerprint analysis, presence of an epithelial marker EpCAM, absence of human papilloma virus (HPV) DNA, and SCC-specific microscopic appearance. Sphere-forming assays and mouse xenografts supported its tumorigenic potential. Spectral karyotype (SKY) analysis revealed numerical and structural chromosomal abnormalities. Whole-exome sequencing (WES) identified 46 non-synonymous and 13 synonymous somatic single-nucleotide polymorphisms (SNPs) and one frameshift deletion in the coding regions. Specifically, mutations of CDKN2A, TP53, SPTBN5, NOTCH2, and FAM136A were found in the databases. Copy number aberration (CNA) analysis revealed that the cell line loses chromosome 3p and 9p, but lacks amplification of 3q and 11q (as does HPV-negative, smoking-unrelated OTSCC). It also exhibits four distinctive focal amplifications in chromosome 19p, containing 131 genes without SNPs. Particularly, 52 genes showed >3- to 4-fold amplification and could be potential oncogenic drivers.(Conclusion) We have successfully established a novel OTSCC cell line from a never-smoking patient. UCSF-OT-1109 is potentially a robust experimental model of OTSCC in never-smokers.

Citation Format: Steven J. Wang, Saurabh Asthana, Annemieke van Zante, Adam B. Olshen, Frank McCormick, Osamu Tetsu. Establishment of an oral tongue squamous cell carcinoma cell line from a never-smoking patient [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3104.
Abstract 3107: Modeling DICER1 syndrome in cells
Authors: Wu Mona K.
Published: August 2018

DICER1 is an endoribonuclease central to generating microRNAs (miRNAs), small RNA molecules that downregulate the expression of approximately 30% of protein-coding genes. Germ-line mutations in DICER1 have been identified in patients afflicted with a pleiotropic tumor predisposition syndrome, usually referred to as DICER1 syndrome (OMIM 606241). DICER1 syndrome is thought to originate from a mesenchymal cell bearing a frameshift or nonsense mutation on one allele and a missense mutation affecting the RNase IIIb domain on the other allele. While interest in the role of DICER1 in normal development and pathogenesis has been explored in both the knockout and overexpression context, neither situation is relevant for understanding DICER1 syndrome. We hypothesize that defective DICER1 protein production in a mesenchymal stem cell at a critical time in development leads to altered miRNA populations that, in turn, initiate or prime cells for tumorigenesis. The goal of this study was to develop and characterize a model of the putative tumor-initiating cell namely a mesenchymal stromal cell (MSC) bearing a DICER1 RNase IIIb mutation. SV-40 immortalized mouse mesenchymal stromal cells (MSCs) containing a homozygous floxed exon for DICER1 were ex vivo cre inactivated to produce Dicer-/- MSCs. Into these Dicer-/- MSCs, a FLAG-tagged human DICER1, a FLAG-tagged RNase IIIa mutant DICER1, and a FLAG-tagged RNase IIIb mutant DICER1 were stably introduced. Cells were characterized by Western blot in response to serum starvation and stimulation. Their ability to form anchorage-independent colonies was assessed by soft agar assay pre- and post-oncogene introduction. Transcriptomes were interrogated by gene expression microarray.

Results: Alterations in protein expression were observed in cells in response to starvation and stimulation. Certain cell lines were able to produce anchorage-independent colonies.

Conclusions: We have created MSCs bearing RNase IIIb-mutated DICER1 to model DICER1 syndrome. We suggest that the ability for an MSC to produce only 3p miRNAs in the presence of an oncogene can allow for anchorage-independent growth.

Citation Format: Mona K. Wu. Modeling DICER1 syndrome in cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3107.
Abstract 3118: Oncogenesis modeling in malignant pleural mesothelioma
Authors: Pruett Nathanael D. , Singh Anand , Bhattacharyya Nisan , Hoang Chuong D.
Published: August 2018

Malignant pleural mesothelioma (MPM) is causally associated with exposure to asbestos and accounts for roughly 75% of mesothelioma cancers. It is an aggressive tumor with a median survival time of approximately 12 months. Treatment options are limited, largely palliative, and still based on cisplatin and pemetrexed, a regimen that remains unchanged since 2003. Despite widespread study, the complexities of MPM biology remain poorly described, especially at the point of tumor initiation where development of novel clinical interventional strategies would be expected to yield the most effect. The extended latency period in humans consisting of decades (over 20-30 years) remains a prohibitive barrier to gain accurate, in vivo insights about MPM oncogenic processes. As such, all in vitro MPM models, which are designed to accelerate tumor development as a practical endpoint of study, to date have not yielded clinically translatable molecular targets. Here, we present a novel in vitro model system for exploring the earliest molecular mechanisms contributing to the development of MPM using normal, human mesothelial cell lines (MeT-5a and LP9-hTERT) engineered to harbor constitutively enhanced NFkB signaling activity. Both 3D-sphere and soft agar assays were used to assess cellular transformation status in these induced cells compared to their wild-type counterparts. Heterotopic xenograft implantation studies were performed in NOD scid-gamma (NSG) immunocompromised mice (sub-cutaneous dorsal flank injection) for tumorigenic potential. In all cases, these induced cells (despite the complete absence of asbestos exposure) exhibited a significant malignant-like phenotype. Tumor growth in mouse xenograft models occurred very rapidly, and was marked by extensive vascular recruitment with invasion into surrounding tissues. In addition, these transformed cells expressed typical mesothelioma markers such as Wilms tumor protein (WT1 gene product) as revealed by immunohistofluorescence. This new MPM model system could represent a powerful construct to explore the early molecular events involved in transformation of normal mesothelial cells leading to malignancy.

Citation Format: Nathanael D. Pruett, Anand Singh, Nisan Bhattacharyya, Chuong D. Hoang. Oncogenesis modeling in malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3118.
Abstract 3120: Tumor-infiltrating lymphocyte profiling in hepatitis B virus pre-S2 mutant-positive hepatocellular carcinoma
Authors: Teng Chiao-Fang , Shyu Woei-Cherng , Jeng Long-Bin
Published: August 2018

Chronic hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC), the leading cause of cancer-related deaths worldwide. Pre-S2 mutant is well demonstrated as a HBV oncoprotein that disturbs multiple cellular signaling pathways, leading to HCC development. HCC patients with pre-S2 mutant are recognized as a high-risk population for postoperative recurrence. Plentiful evidences reveal that tumor infiltrating lymphocytes (TILs) play an essential role in the stepwise progression of HCC development, and are closely associated with clinical outcomes. However, to date, the role of pre-S2 mutant in regulation of TILs distribution in HCC remains totally unknown. In this study, we collected plasma and paraffin-embedded liver samples from HBV-related HCC patients. The plasma samples were used for detection of the pre-S2 mutant with polymerase chain reaction-based method. As a result, the patients were divided into two groups: the pre-S2 mutant-positive and -negative groups. The paraffin-embedded liver samples were used for examination of the distribution of specific population of TILs with fluorescent immunohistochemistry staining. We analyzed 6 cases of HBV-related HCC samples, half of which were detected as pre-S2 mutant-positive. The distribution level of the cytotoxic CD3+CD8+ TILs in HCCs was examined. We found that patients with pre-S2 mutant had a considerably lower density of cytotoxic CD3+CD8+ TILs in HCC tissues than patients without pre-S2 mutant. The mean ± SEM of cytotoxic CD3+CD8+ TILs density was 3.74 ± 0.60 and 6.66 ± 0.66 in patients with and without pre-S2 mutant, respectively (p value=0.031). Our result suggests that pre-S2 mutant may have a role in regulation of TILs distribution in HBV-related HCCs. Systematic evaluation of specific population of TILs may help guide development of novel strategies for prognosis and/or therapies of HCC, especially for the HBV-related pre-S2 mutant-positive high-risk patient population.

Citation Format: Chiao-Fang Teng, Woei-Cherng Shyu, Long-Bin Jeng. Tumor-infiltrating lymphocyte profiling in hepatitis B virus pre-S2 mutant-positive hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3120.
Abstract 3124: Cytotoxic effects of tumor-specific T lymphocytes on autologous cancer initiator stem-like glioblastoma cells
Authors: Huang Yin-Cheng , Wei Kuo-Chen , Chang Chen-Nen , Yang Wen K.
Published: August 2018

[Purpose] In clinical trial of autologous dendritic cell/autologous tumor cell antigens (ADCTA) adjuvant immunotherapy of glioblastoma multiforme (GBM), we studied if and how GBM CD133(+) stem-like initiator cells are susceptible to immune cytotoxic eradication

[Methods] Tumor-infiltrating T lymphocytes (TILs) were isolated from GBM patients after the ADCTA vaccination and tested for their effects on CD133(+) and CD133(-) glioma cells separated from the same patients.

[Results] TILs isolated from GBM patients after ADCTA vaccination were cytotoxic to both CD133(+) and CD133(-) autologous glioma cells. However, the cell lysis was detectable in CD133(-) in 4 hours but did not occur until more than 18 hours later in the CD133(+) cancer stem-like initiator cells. Surprisingly, we found that, upon exposure to the TILs, the GBM tumor cells showed phenotype shift from CD133(+) to CD133 (-). Similar CD133(+) to CD133(-) phenotype shift was also observed by exposure to culture condition medium of the TILs. The responsible factors secreted by the TILs that caused the phenotype shift was determined to be INFγ and TNFα.

[Conclusion] ADCTA vaccination in GBM patients would generate TILs that are not only specifically cytotoxic to autologous CD133(-) glioma cells but also secret INFγ and TNFα etc to induce phenotypic shift of the resistant CD133(+) stem-like GBM to susceptible CD133(-) glioma cells.

Citation Format: Yin-Cheng Huang, Kuo-Chen Wei, Chen-Nen Chang, Wen K. Yang. Cytotoxic effects of tumor-specific T lymphocytes on autologous cancer initiator stem-like glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3124.
Abstract 3127: Peripherally induced Tregs or pTregs are the potent tolerance inducer for the growth and metastasis of cancer
Authors: Hegde Upendra P. , Chakraborty Nitya G.
Published: August 2018

Regulatory T cells act as the most important tolerance inducer for the growth and metastasis of cancer. It remains a wonder how Tregs induce tolerance for the development of cancer. Previously we have shown with melanoma patients that, increase in pTreg cell number in blood are related to the poor prognosis of the disease. pTreg and iTreg are remarkably similar and significantly different in functionality from tTreg. Here, we worked with four melanoma patients two HLA A2 + and two HLA A2 negative. PBL and tumor cells were obtained from the patients with informed consent. Treg cells were generated in four different culture conditions: isolated from tumor +PBL IVC or peptides, Mart-1 A2 or Flu A2 pulsed DC + PBL IVC, with purified CD4 cells stimulated with anti CD3 and antiCD28 plus IL-2 with or without TGF-b. Here we show some phenotypic and functional characteristics of the induced Treg cells in cultures from the patients' PBL derived CD4+CD5- cells and analyzed those in separate CTL generation assays. We observed that induced Treg cells under different conditions do not uniformly express CD25, FoxP3, PDL-1 or CTLA 4 as the known surface markers. When analyzed for their functionality, with adjusted number of cells, in suppressing the anti tumor CTL response, a significant difference was observed. The most effective Tregs cells were found to be those isolated from autologous tumor +PBL IVC or from Mart-1 peptide pulsed IVC. Those cells completely blocked the CTL induction and secreted huge amount of IL-10 upon re-stimulation. Further analysis with these different types of induced Treg cells in terms of various gene expressions will be useful to find a target molecule to block such expansion of pTregs cells for better therapeutic outcome.

Citation Format: Upendra P. Hegde, Nitya G. Chakraborty. Peripherally induced Tregs or pTregs are the potent tolerance inducer for the growth and metastasis of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3127.
Abstract 3136: Comprehensive profiling the immune-status of a broad range of in vivo syngeneic models
Authors: Huang Jingqi , Hu Shipzong , Li Wentao
Published: August 2018

Latterly with approval of PD-1, PD-L1 antibodies in clinical oncology, immunotherapy has gain recognition for cancer treatment for changing the way by joining chemotherapy, radiation and surgery. This type of cancer treatment aims to produce greater effectiveness with less toxicity than cancer chemotherapy and mainly acts by boosting the body's natural defenses to fight the cancer. It uses substances made by the body or laboratory agent to improve or restore the function of immune system. Therefore, understood the nature immune status and micro environment of in vivo tumor models is very important to assist exploring immunotherapy. As a results, nearly 40 tumor cell lines over a broad range of tumor type as well as corresponding in vivo syngeneic models were intensively studied on its immune status under two conditions when tumor volume were 100 mm3 and 500 to 600mm3 . The effort has been focus on the immune status including T cell status and levels of immune-suppression via FACS analysis of the population of CD4+, CD8+, Treg, MDSC, macrophage and DC cells. Furthermore, not only population of immune cells but also the expressions of immune related gene were analyzed by RE-PCR which are the genes related regulatory of T cells i.e., CD4, Foxp3, GITR; gene of tumor associated macrophage CSF1R, CD68, CX3CR1, ITGAM, CD163, MARC1; gene of co-stimulator receptors of T cells ICOS, CD137(Tnfrsf9), OX40(Tnfrsf4); co-inhibitory receptors of T cell BTLA, HAVCR2, CTLA4, LAG3, CD28, PDCD1; and gene of co-inhibitory ligands of tumor cells CD274, CD137L(Tnfsf9), PDCD1LG2, HVEM(Tnfsf14), OX40L(Tnfsf4). The profiling data illustrated, indeed, the expression of PD-L1 is different on different tumor cells; the immune status certainly pending on the tumor type but also related to the tumor progression. The bigger tumor size the higher level of expression of immune suppression which deterring the immune cell ability to conquer cancer. We believe that this profiling data will help many scientists to properly select correct model to support R&D and to better understand how immune therapeutically agent acts in the immune system. With this comprehensive research data, we're now at a point where this information has started to become translated into treatment possibilities for cancer patients.

Citation Format: Jingqi Huang, Shipzong Hu, Wentao Li. Comprehensive profiling the immune-status of a broad range of in vivo syngeneic models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3136.
Abstract 3148: p140Cap affects breast cancer features through the regulation of the tumor microenvironment composition
Authors: Salemme Vincenzo
Published: August 2018

Increasing data indicate the key role of the tumor microenvironment in cancer progression, metastasis formation and treatment sensitivity, where the tumor stroma and the immune infiltrate strongly influence tumor features. Our recent studies indicate that the adaptor protein p140Cap is highly expressed in about 50% of ERBB2-amplified breast cancer patients. Its expression correlates with increased patient survival and decreased probability to develop metastasis. Causally, p140Cap negatively regulates breast tumor properties both in vitro and in the NeuT preclinical mouse model. In this study, we observed an indirect influence of p140Cap-expression on tumor immune infiltrate composition of a syngeneic ERBB2+ breast cancer model. p140Cap tumor cells and tumors show reduced level of G-CSF mRNA and protein. Accordingly, we observed a reduced mobilization of G-CSF-dependent myeloid-derived suppressor cells (MDSCs- CD11b+Ly6G+Ly6Clo) from bone marrow and a decreased level of MDSCs in tumor, blood and spleen of mice orthotopically injected with p140Cap tumor cells, compared to controls. This “less” immune suppressive microenvironment associated to p140Cap-expression, was also confirmed by an increase in CD8+, M1 macrophages and Natural Killer lymphocytes concomitant with a decrease in M2 macrophages and FoxP3 regulatory T cells. Interestingly, the expression of p140Cap in cells and tumors significantly reduce the fraction of cancer stem cells and their sphere and tumor initiation frequency, by modulating their proliferative potential. Since that the tumor initiating cell exhibit an enhanced G-CSF production, these data suggest that p140Cap, by regulating the cancer stem cell compartment, limits ERBB2 tumor aggressiveness by creating a tumor microenvironment less favorable to tumor progression.

Citation Format: Vincenzo Salemme. p140Cap affects breast cancer features through the regulation of the tumor microenvironment composition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3148.
Abstract 3150: Serglycin promotes cell migration mediated through the interaction of its GAG moiety with CD44
Authors: Chen Joanne Jeou-Yuan , Guo Jing-You , Chiu Chu-Hsuan , Wang Mei-June
Published: August 2018

Tumor microenvironment (TME) plays an important role in cancer progression. We have recently shown that serglycin (SRGN), a chondroitin sulfate proteoglycan (CSPG), is overexpressed in primary non-small cell lung cancers (NSCLCs), by carcinoma as well as stromal cells, and that the secreted SRGN can bind to CD44 and promotes NSCLC aggressiveness (Oncogene 36:2457, 2007). In this study, we further examined the molecular mechanisms underlying SRGN-elicited cell migration. By gain- and loss-of-function studies, we first showed that SRGN promoted cell migration in a CD44-dependent manner. SRGN promoted SRC activation, leading to increased phosphorylation of paxillin (PAX) at Y118 as well as reduced PAX/FAK complex formation, suggesting that SRGN promotes cell migration through facilitating focal adhesion turnover. In support, knockdown of SRC or treatment of cells with PP2, a SRC family inhibitor, abolished SRGN-elicited migratory activity. In addition, we showed that SRGN induced actin cytoskeleton reorganization and promoted lamellipodia and filopodia formation at the leading edge, facilitating a directional movement during wound closure in NSCLC cells. In consistence, increased levels of activated RAC1, which is required for lamellipodia formation, and CDC42, which is required for filopodia formation, were detected. We further examined the role of CS modification in SRGN-mediated function. We generated the SRGN(S/A) mutant in which the eight serine residues involved in GAG modification were converted to alanine, and showed that SRGN(S/A) mutant not only failed to induce the phosphorylation of SRC and paxillin, but also failed to promote cell migration. In consistence, pre-treatment of SRGN-containing conditional medium with Chondroitinase ABC also suppressed SRGN-elicited cell migration. Taken together, our data demonstrated that SRGN interacts with CD44 through its GAG modifications, leading to increased cell migration by promoting FA turnover and cytoskeleton reorganization.

Citation Format: Joanne Jeou-Yuan Chen, Jing-You Guo, Chu-Hsuan Chiu, Mei-June Wang. Serglycin promotes cell migration mediated through the interaction of its GAG moiety with CD44 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3150.
Abstract 3155: Loss of YAP1 dysregulates RhoA signaling and promotes cell invasion
Authors: Hendrick Natalie M. , Iwanicki Marcin P. , Li Carman Man-Chung , Bui Duyen Amy , Selfors Laura M. , Brugge Joan S.
Published: August 2018

While Hippo pathway signaling undoubtedly plays an important role in cancer, the role of its effector YAP1 as an oncogene or tumor suppressor is context dependent. The precise conditions that support the tumor-promoting or tumor-suppressing roles of YAP1 remain largely undefined. While YAP1 can drive oncogenic programs through its nuclear function as a transcriptional coactivator of TEAD and other transcription factors, previous findings from our lab and others suggest that loss of YAP1 function can also have tumor promoting consequences. One well-characterized role of YAP1 is that of a nuclear relay of signals triggered by mechanical forces that lead to activation of Rho GTPases. Our new studies using a combination of genetic, biochemical, and microscopy techniques provide evidence that YAP1 is not only activated by Rho, but can also regulate Rho activation and the actin cytoskeleton. Knockdown of YAP1 in MCF10A immortalized mammary epithelial cells resulted in increased lamellipodia formation in culture as seen by time-lapse microscopy, and increased protrusive, invasive activity in 3D Matrigel culture. YAP1 knockdown also induced a scattering phenotype, characterized by cell dissociation from the leading edge of a wounded monolayer. While E-cadherin protein levels were unaffected by YAP1 knockdown, adherens junction maturation at cell-cell junctions was compromised. These morphological alterations were associated with decreased levels of active RhoA, as well as a decrease in downstream myosin light chain phosphorylation. Our results indicate loss of YAP1 decreases active RhoA, leading to reduced cell-cell adhesion and increased protrusive cellular behavior. Our findings raise the possibility that YAP1 is not only a Rho-sensor, but also mediates a feedforward regulatory loop to maintain Rho-regulated cytoskeletal structures, and that loss of YAP1 through genetic deletion in tumor cells could promote invasive behavior.

Citation Format: Natalie M. Hendrick, Marcin P. Iwanicki, Carman Man-Chung Li, Duyen Amy Bui, Laura M. Selfors, Joan S. Brugge. Loss of YAP1 dysregulates RhoA signaling and promotes cell invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3155.
Abstract 3160: The actin-binding protein Girdin/GIV regulates collective cancer cell migration by controlling cell adhesion and cytoskeletal organization
Authors: Wang Xiaoze , Enomoto Atsushi , Weng Liang , Haga Hisashi , Ishida Sumire , Takahashi Masahide
Published: August 2018

Pathological observations show that cancer cells frequently invade the surrounding stroma in collective groups rather than through single cell migration. The identification of genes and proteins that are specifically involved in the collective behavior of cancer cells has thus far been limited. Here, we studied the role of the actin-binding protein Girdin in collective cancer cell migration. This protein is a specific regulator of collective migration of neuroblasts born in the subventricular zone of the postnatal and adult brains and participates in the progression of cancer. We found that Girdin was essential for the collective migration of the skin cancer cell line A431 on collagen gels as well as their fibroblast-led collective invasion in an organotypic culture model. We provide evidence that Girdin binds to β-catenin that plays important roles in the Wnt signaling pathway and in E-cadherin-mediated cell-cell adhesion. Girdin-depleted cells displayed scattering and impaired E-cadherin-specific cell-cell adhesion. Importantly, Girdin depletion led to impaired cytoskeletal association of the β-catenin complex, which was accompanied by changes in the supracellular actin cytoskeletal organization of cancer cell cohorts on collagen gels. Although the underlying mechanism is unclear, this observation is consistent with the established role of the actin cytoskeletal system and cell-cell adhesion in the collective behavior of cells.

Citation Format: Xiaoze Wang, Atsushi Enomoto, Liang Weng, Hisashi Haga, Sumire Ishida, Masahide Takahashi. The actin-binding protein Girdin/GIV regulates collective cancer cell migration by controlling cell adhesion and cytoskeletal organization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3160.
Abstract 3161: The macrolide toxin mycalolide B disrupts actin-driven invasion and metastasis of HER2-positive cancers
Authors: Nersesian Sarah , Williams Rodette , Evans Dr. Andrew , Allingham Dr. John , Craig Dr. Andrew
Published: August 2018

HER2 is a driver and clinical target in cancers afflicting women. HER2-positive (HER2+) cancers have high rates of metastasis and lower overall survival rates compared to other cancer subtypes. The goal of this study is to test the vulnerability of HER2+ cancer cells and tumors to disruption of their actin cytoskeleton using the marine macrolide toxin mycalolide B (Myc B), as these cells are highly dependent on rapid actin polymerization and remodeling for migration and invasion into new sites of tumor growth. The effects of Myc B treatment on HER2+ breast (HCC1954) and ovarian (SKOV3) cancer cell lines were profiled in assays of cell viability, motility, and invasion. Treatments of Myc B alone or in combination with Trastuzumab were also performed in HER2+ tumor xenograft assays. Myc B showed potent growth suppressive and cytotoxic effects on HER2+ cancer cells at doses in the 70-100 nM range. At sub-lethal doses, Myc B caused a rapid loss of leading edge protrusions, and sustained defects in HER2+ cancer cell motility and invasion. HER2 internalization and killing of HER2+ cancer cells by Trastuzumab-emtansine was not compromised with Myc B treatment. In a HER2+ tumor xenograft model, Myc B treatment alone, or in combination with Trastuzumab, led to significant reductions in tumor growth and metastasis. Together, these findings identify a major vulnerability in metastasis-initiating HER2+ cancer cells to the actin toxin Myc B, and provide a rationale to exploit this vulnerability with the development of new therapeutics targeting actin.

Citation Format: Sarah Nersesian, Rodette Williams, Dr. Andrew Evans, Dr. John Allingham, Dr. Andrew Craig. The macrolide toxin mycalolide B disrupts actin-driven invasion and metastasis of HER2-positive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3161.
Abstract 3171: Vangl2 regulates cancer stem cell self-renewal and growth in rhabdomyosarcoma
Authors: Hayes Madeline , McCarthy Karin , Jin Alexander , Iyer Sowmya , Garcia Sara , Oliveira Mariana L. , Sindiri Sivasish , Gryder Berkley , Motala Zainab , Nielsen G Petur , Borg Jean-Paul , Rijn Matt van de , Malkin David , Khan Javed , Ignatius Myron , Langenau David M.
Published: August 2018

Growth and relapse are driven by cancer stem cells (CSCs) in a subset of tumors, yet mechanisms driving cancer cell fate choices, maintenance and self-renewal are not fully understood. Here, we show that Van Gogh-like 2 (Vangl2), a core regulator of the non-canonical Wnt/planar cell polarity pathway (Wnt/PCP), regulates CSCs self-renewal in human rhabdomyosarcoma (RMS) – a common pediatric cancer of muscle. Wnt/PCP signaling is essential during development and recent work has linked this pathway to cancer growth, invasion and metastasis. However, roles for Vangl2 in regulating tumor self-renewal have not been previously described. Here, we show that VANGL2 is expressed in a majority of human RMS, specifically within early mononuclear progenitor-like cells. VANGL2 depletion inhibited proliferation, reduced self-renewal, and induced differentiation of human RMS. VANGL2 was also required for continued tumor growth and maintenance following engraftment of human RMS using mouse xenografts. Using a zebrafish model of embryonal rhabdomyosarcoma (ERMS) and limiting dilution cell transplantation approaches, we identified that Vangl2 expression enriches for CSCs in vivo and when transgenically expressed, at high levels elevates cancer stem cell number by 9-fold. Mechanistic studies revealed a role for RhoA downstream of Vangl2 in regulating maintenance of stem cell programs in human RMS. Our studies offer novel opportunities to isolate and characterize RMS cancer stem cells in vivo, and identify potential therapeutic targets for patient treatment.

Citation Format: Madeline Hayes, Karin McCarthy, Alexander Jin, Sowmya Iyer, Sara Garcia, Mariana L. Oliveira, Sivasish Sindiri, Berkley Gryder, Zainab Motala, G Petur Nielsen, Jean-Paul Borg, Matt van de Rijn, David Malkin, Javed Khan, Myron Ignatius, David M. Langenau. Vangl2 regulates cancer stem cell self-renewal and growth in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3171.
Abstract 3197: Beta-Apopicropodophyllin, a derivative of podophyllotoxin, has a function as anticancer drug and radiosenstizer against non-small cell lung cancer cells
Authors: Kim Ju Yeon , Cho Jeong Hyun , Hwang Sang-Gu , Song Jie-Young , Lee EunAh , Um Hong-Duck , Park Jong Kuk
Published: August 2018

We have synthesized new 32 derivatives of podophyllotoxin and screened candidate showed anti-cancer effect. Among these derivatives, we found beta-Apopicropodophyllin (APP) is candidate derivative inducing cell death against non-small cell lung cancer (NSCLC) cell lines: A549, NCI-1299 and NCI-460 (IC50 values =16.9, 13.1 and 17.1 nM, respectively). We also analyzed MOA (mode of action) of APP and found APP induced cell death via various mechanisms. Firstly, APP inhibits the polymerization of microtubule and induces G2/M phase arrest as well as DNA damage. APP treatment induces expressions of mitosis-related proteins, phospho-ATM, γ-H2AX and p21. Next, the intrinsic and extrinsic apoptotic pathways by APP are activated as shown in the activations of caspase-3, -8 and -9. APP also induces pro-apoptotic ER stress signaling pathway as indicated by elevated expression levels of BiP, phospo-eIF2α, CHOP and ATF4. Additionally, APP also showed radiosensitizer function. Combination of APP and gamma-ionizing radiation (IR) enhanced apoptotic cell death of NSCLC cell via amplification of ER stress. Taken together, these results implied APP might be a potent anti-cancer drug candidate inducing microtubule disruption, DNA damage, cell cycle arrest and ER stress.

Citation Format: Ju Yeon Kim, Jeong Hyun Cho, Sang-Gu Hwang, Jie-Young Song, EunAh Lee, Hong-Duck Um, Jong Kuk Park. Beta-Apopicropodophyllin, a derivative of podophyllotoxin, has a function as anticancer drug and radiosenstizer against non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3197.
Abstract 3201: A theranostic approach using photodynamic therapy to efficiently kill neuroblastoma cancer cells through induction of apoptosis
Authors: Davids Lester M. , Schwar Anja , Lang Dirk
Published: August 2018

Neuroblastoma is an extra-cranial tumour of childhood and arises from the sympatho-adrenal lineage of the neural crest tissue. Despite advances in treatment strategies, significant complications in curing this cancer remain. Furthermore, severe consequential effects on patient health have been associated with chemotherapy and/or radiation. Photodynamic therapy (PDT) with the photosensitiser hypericin (HYP) presents an attractive and efficient adjunctive therapy alternative for this aggressive cancer. HYP, an extract from Hypericum perforatum (St John's Wort) species, has a high quantum yield following photo-activation and a low dark toxicity and genotoxiciy. This study tests the anti-cancer efficacy of HYP-PDT in SK-N-SH neuroblastoma cells using an in vitro tissue culture model. Results show through detailed uptake kinetics that HYP is absorbed effectively by SK-N-SH cells after 6 hours of incubation and live fluorescent confocal microscopy revealed primary localisation of HYP to lysosomes and mitochondria. Through cell viability assays it was determined that 2, 3 and 4μM HYP-PDT resulted in 45.6±5.1% cell death within 24 hours of treatment. Additionally, 24 hours post 0.25μM and 2μM HYP-PDT, we observed distinct morphological changes in SK-N-SH cells using phase contrast microscopy, and determined a high apoptotic mode of cell death using Fluorescent Activated Cell Sorting (FACS). These results demonstrate that HYP-PDT is effective in killing neuroblastoma cells and that the mode of cell death is apoptotic in nature. This study is ongoing with further investigation into the signalling underlying this photo-toxicity with particular emphasis on cell death resistance mechanisms in neuroblastoma cells.

Citation Format: Lester M. Davids, Anja Schwar, Dirk Lang. A theranostic approach using photodynamic therapy to efficiently kill neuroblastoma cancer cells through induction of apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3201.
Abstract 3207: CXCR4 endows radioresistance in non-small cell lung cancer cells through STAT3-mediated Slug expression
Authors: Lee Dae-Hee , Kim Jeong-Yub , Kim Hee-Jin , Park Myung-Jin
Published: August 2018

Lung Cancer is one of the most common causes of cancer-related mortality worldwide, despite of significant advance of treatment strategies including chemotherapy and radiotherapy. Although chemotherapy is the first choice for treatment of non-small cell lung cancer cells (NSCLCs) except surgery, radiotherapy is primary treatment modality for patients with unresectable NSCLC on its own or with another treatment like chemotherapy. CXCR4 is chemokine (C-X-C motif) receptor 4 whose ligand is Stromal-derived-factor-1 (SDF1a), so-called CXCL12. According to the current reports, CXCR4 is widely expressed by malignant tumor and crucial for tumor growth, vascularization, metastasis, cell migration and related to poor prognosis. Here, we show that CXCR4 signaling confers resistance to ionizing radiation (IR) in NSCLCs. Drug-resistant A549/GR cells with enhanced CXCR4 expression exhibited high colony forming ability after IR compared with A549 cells. Transfection of siRNA against CXCR4 or treatment of pharmacological inhibitor (AMD3100) sensitized these cells to IR. Reversely, overexpression of CXCR4 in A549 and H460 enhanced clonogenic survival after IR. IR stimulated CXCR4 expression and CXCR4-mediated STAT3 phosphorylation in NSCLCs. Inhibition of STAT3 by WP1066 or siSTAT3 significantly suppressed the colony forming ability, indicating that CXCR4-mediated STAT3 signaling is important for IR resistance in NSCLC cells. CXCR4/STAT3 signaling upregulated the expression of Slug and down-regulation of Slug with siRNA increased IR sensitivity in NSCLCs. Taken together, our data suggest that CXCR4/STAT3/Slug axis is crucial for IR resistance of NSCLCs and is able to be considered as a therapeutic target for enhancing IR sensitivity of this devastating cancer.

Citation Format: Dae-Hee Lee, Jeong-Yub Kim, Hee-Jin Kim, Myung-Jin Park. CXCR4 endows radioresistance in non-small cell lung cancer cells through STAT3-mediated Slug expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3207.
Abstract 3213: Evaluation of porphyrin biodistribution for use in photodynamic therapy of retinoblastoma in transgenic mice
Authors: Poyer Florent , Hugeut Samuel , Lemaitre Stéphanie , Madar Olivier , Thomas Carole D , Maillard Philippe , Teulade-Fichou Marie-Paule , Cassoux Nathalie , Doz François , Rezai Keyvan
Published: August 2018

Background: Porphyrins are a group of heterocyclic macrocycle organic compounds. They have shown good efficiency in the photodynamic treatment of tumors in the last twenty years. The photodynamic therapy could be an alternative treatment for retinoblastoma using a photosensitizer with low dark toxicity and low mutagenic properties unlike the conventional chemotherapies. The objectives of this study were to develop a photosensitizer in our lab which is a diethylene glycol mannoconjugated tetraphenyl porphyrin (TPP, MW: 1413.6) and to determine the optimal interval time for photodynamic therapy using the concentration of TPP in eye and blood samples of mice. Materials and method: Transgenic mice developed bilateral retinoblastoma from the retina since 4 weeks of age to 16 weeks of age (tumors fill the ocular globe and the mice will be sacrificed for ethic purpose). The evaluation of the biodistribution of our TPP was realized to know the best drug-light interval to perform the photodynamic therapy. For this purpose, an intraperitoneal injection of 200 µl at 5.25 mg/ml in PEG 400/physiological serum (1:1, v/v) was performed on the LHbetaTag transgenic mice. At different time intervals (8h, 24h, 40h, 48h, and 72h) following injection, the mice were sacrificed and the eyes and blood were collected. TPP in eye samples were extracted in methanol and the level of TPP was measured by ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) analysis (0.5-100 ng/mL). The level of TTP in blood samples was quantified after protein precipitation by UPLC-MS/MS method (5.00-1000 ng/mL).

Results: We observed a time dependency accumulation of TPP in eyes with observed Tmax=48 hours. TPP serum concentrations decreased by the same manner up to 48 h. The determination of drug-light interval allowed us to perform the light delivery when the TPP level is highest (48 h) in the ocular tumor tissue (retinoblastoma).

Conclusions: Our TPP had a good in vitro photocytotoxicity at very low doses and showed a good efficacy in vivo with subcutaneous patient derived retinoblastoma xenografts in immunodeficient mice. The knowledge of Tmax and this interval is essential to realize the photodynamic therapy with the better efficiency, the final goal being to eradicate the retinoblastoma from the retina.

Citation Format: Florent Poyer, Samuel Hugeut, Stéphanie Lemaitre, Olivier Madar, Carole D Thomas, Philippe Maillard, Marie-Paule Teulade-Fichou, Nathalie Cassoux, François Doz, Keyvan Rezai. Evaluation of porphyrin biodistribution for use in photodynamic therapy of retinoblastoma in transgenic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3213.
Abstract 3214: FePt nanoparticles overcoming radiotherapy resistance in hCtr-1 high expression cells via mitochondria targeting
Authors: Tsai Tsung-Lin , Kuo Macus Tien , Wang Jen-Ya , Shin Wan-Li , Chen Helen H. W. , Su Wu-Chou
Published: August 2018

The glutathione (GSH) overexpression small-cell-lung-cancer cell lines, SR3A-13 and SR3A-14, transfected with recombinant DNA encoding γ-glutamylcysteine synthetase (γ-GCSh), are radiation resistant. However, they are more sensitive to cisplatin treatment, compared with parental cell (SR3A)1. In addition to be a copper transporter, the hCtr-1 is also a transporter for platinum (Pt)-based agents. Therefore, the cellular uptake of cisplatin is enhanced by the upregulation of human copper transporter 1 (hCtr-1) at the cell membrane. We have synthesized an alloy FePt nanoparticles (FePt NPs) that generated excellent contrast effects in both CT and MRI images2. Interestingly, the uptake of FePt NPs by SR3A-13 and -14 cells was 3-3.5 fold higher than that of SR3A cells, measured with inductively coupled plasma optical emission spectrometry (ICP-OES) and transmission electron microscopy (TEM). Because intracellular NPs have been shown to generate reactive oxygen species (ROS) and overproduction of ROS may enhance the efficacy of radiotherapy, we sought to explore whether treatment with the alloy FePt NPs can reverse the radiation resistance in SR3A-13 and -14 cells. We found co-treatment with the alloy FePt NPs and radiation effectively reduce the survivals of these 2 cells. Furthermore, we found an alternation in mitochondrial appearance accompanied with low signal of Tetramethylrhodamine, methyl ester (TMRM), indicating a malfunction is induced in mitochondria. We conclude that FePt NPs are able to overcome radiation resistance in hCtr-1 high expression cells via mitochondria targeting.

Citation Format: Tsung-Lin Tsai, Macus Tien Kuo, Jen-Ya Wang, Wan-Li Shin, Helen H. W. Chen, Wu-Chou Su. FePt nanoparticles overcoming radiotherapy resistance in hCtr-1 high expression cells via mitochondria targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3214.
Abstract 3225: Cutaneous nevi and cancer risk among US women
Authors: Li Xin , Wu Wenting , Giovannucci Edward , Stampfer Meir J. , Gao Xiang , Han Jiali
Published: August 2018

Background: Longer telomeres have been linked to elevated cutaneous nevus number. Recently, a large Mendelian randomization study identified a positive association between telomere length and risk of cancer. We hypothesized that higher nevus count, as a phenotypic marker of longer telomere length, may be associated with increased risk of cancer development.

Methods: We prospectively examined the association between nevus count and total as well as site-specific cancer risk among participants in the Nurses' Health Study (NHS, 1986-2012) and the Nurses' Health Study 2 (NHS2, 1989-2013).

Results: During 3,941,477 person-years of follow-up, we documented a total of 24,428 cancer cases (16,297 in the NHS and 8,131 in the NHS2). Compared to participants who had no nevi, the multivariate HRs were 1.08 (95%, 1.05-1.11) for women with 1-5 nevi, 1.15 (95%, 1.09-1.21) for those who had 6-14 nevi, and 1.26 (95%, 1.18-1.34) for those with 15 or more nevi (p trend < 0.0001). We did not observe significant interactions between the number of nevi and other covariates in the multivariable-adjusted model. We also conducted secondary analyses by excluding 1) only melanoma and 2) both melanoma and breast cancer from the outcomes of interest, respectively. The results were very similar to those of our primary analysis using all cancer sites. For individual cancer sites, most of the associations with mole counts were positive but not statistically significant.

Conclusion: We identified the number of cutaneous nevi as a phenotypic marker associated with cancer risk, which may possibly be explained by telomere biology.

Citation Format: Xin Li, Wenting Wu, Edward Giovannucci, Meir J. Stampfer, Xiang Gao, Jiali Han. Cutaneous nevi and cancer risk among US women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3225.