bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1897: Determining the role of PRCP/PREP in triple negative breast cancer
Authors: Perez Ricardo E. , Duan Lei , Maki Carl G.
Published: August 2018

Triple negative breast cancer (TNBC) accounts for 15-20% of all breast cancer cases. Currently, the only effective treatment for TNBC is chemotherapy agents. A well-established characteristic of TNBC is the ability of these cells to become metastatic following chemotherapy treatment resulting in increased mortality. New forms of treatment which target TNBC are urgently needed. Insulin receptor substrate 1 (IRS1) has been extensively studied in the regulation of the insulin and insulin-like growth factor receptor signaling cascades, specifically in the induction of the intracellular PI3K/AKT/mTORC1 and MAP kinase pathways. The roles of such pathways in cancer are currently being studied and considered for therapeutic targeting. We previously showed that the stability and levels of IRS1 are maintained by prolylcarboxypeptidase proteins PRCP/PREP in pancreatic cancer cells. Our hypothesis is that PRCP/PREP are potential therapeutic targets in TNBC. Specifically, we hypothesize that blocking the expression or reducing the activity of PRCP/PREP will inhibit the PI3K/AKT/mTORC1 pathway in TNBC cells and, in turn, reduce growth and survival in these cells. To test this, we will be treating a number of TNBC cell lines with a PRCP/PREP inhibitor and determining IRS1/2 protein levels, AKT/mTORC1 signaling, and growth and survival. TNBC cells were screened for sensitivity against the PRCP/PREP inhibitor, Y-ox. Sensitive cells treated with Y-ox showed earlier reduction in the IRS1/AKT/mTORC1 signaling, as well as earlier loss of cell viability compared to those cells with relative resistance to Y-ox. Similarly, cells treated with the IRS1/2 inhibitor NT-157 and the AKT inhibitor, MK-2206 showed loss of cell viability similarly seen with the PRCP/PREP inhibitor, Y-ox. In conclusion, the results shown in this study show a possible new therapeutic target against triple negative breast cancer in the inhibition of PRCP/PREP leading to loss of cell proliferation and viability.

Citation Format: Ricardo E. Perez, Lei Duan, Carl G. Maki. Determining the role of PRCP/PREP in triple negative breast 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 1897.
Abstract 1904: Polyprenoic acid, the first-discovered hepatocyte nuclear factor 4 alpha agonist, inhibits multistep liver carcinogenesis
Authors: Yamashita Taro , Okada Hikari , Honda Masao , Kaneko Shuichi
Published: August 2018

Hepatocyte nuclear factor 4 alpha (HNF4α) is one of the most ancient nuclear receptors known as a master regulator of hepatocytes in lipids, carbohydrates, and drug metabolism. It also works as a tumor suppressor to regulate cell cycle in hepatocellular carcinoma (HCC), but the ligand activating HNF4α has not yet been discovered thus far. Here we evaluated the effect of polyprenoic acid (PA), formerly known as an acyclic retinoid, on HNF4α activation in dysplastic nodule and HCC in vitro and in vivo. PDGF-C transgenic (Tg) mice was evaluated by Gd-EOB-DTPA-enhanced MRI to examine the effect of PA on HCC development in vivo. Whole exome sequence analysis was performed using Illumina Hiseq 2000 system. Contrast enhanced CT scan data of patients enrolled in previous phase II/III randomized placebo-controlled study were collected and analyzed (n = 124 for PA and 127 for placebo). PA treatment regressed the liver dysplastic nodule and a subset of HCCs in PDGF-C Tg mice evaluated by MRI. This tumor regression was accompanied with the reduction of accumulated gene mutations, inactivation of cell cycle, and transcriptional activation of HNF4α-target genes. The binding of PA on ligand binding domain of HNF4α was shown by docking simulation in silico and immunoprecipitation-mass spectrometry analysis in vitro. Transcriptional activation of HNF4α by PA was also verified using a luciferase reporter assay and binding of HNF4α on DNA binding elements. PA treatment in HCC cells immediately resulted in the degradation of HNF4α by ubiquitin-proteasome system with activation of DNA damage responses when cultured in lipid depleted medium. In vivo knockdown of HNF4A cancelled the effect of PA on regression of liver tumor growth in PDGF-C Tg mice. PA treatment (600 mg/day) suppressed the HCC development from dysplastic nodule compared with placebo in human with statistical significance (HR 0.41; 95% CI, 0.17~0.98, P = 0.046). Taken together, our data indicated that PA is the first-discovered HNF4α ligand to activate its function. PA activates the DNA damage responses and suppresses the accumulation of gene mutations especially in dysplastic nodule, warranting the future prospective study to evaluate the effect of PA on suppression of HCC development from dysplastic nodules in human.

Citation Format: Taro Yamashita, Hikari Okada, Masao Honda, Shuichi Kaneko. Polyprenoic acid, the first-discovered hepatocyte nuclear factor 4 alpha agonist, inhibits multistep liver carcinogenesis [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 1904.
Abstract 1915: Validation of ero1l as a therapeutic target for the treatment of multiple myeloma
Authors: Chen Weichih , Geldenhuys Werner , Hazlehurst Lori
Published: August 2018

Validation of ERO1L as a therapeutic target for the treatment of multiple myeloma

Wei-Chih Chen1, Werner Geldenhuys1 and Lori Hazlehurst1,2,3

1. Department of Pharmaceutical Sciences, School of Pharmacy.

2. Osborn Hematopoietic Malignancy and Transplantation Program, West Virginia University.

3. West Virginia University Cancer Institute, Morgantown, WV, USA.

Multiple myeloma (MM) is a hematologic malignancy of plasma cells and remains an incurable disease. MM is exquisitely sensitive to agents which induce ER stress including inhibitors that target the proteasome such as Bortezomib. However, MM patients eventually relapse with drug resistant disease, indicating that additional therapies are required to improve patient outcomes. Endoplasmic reticulum oxidase 1-alpha (ERO1L) is an endoplasmic reticulum (ER) resident protein that plays a critical role in disulfide bonds formation and contributes to proper folding of secretory proteins. High expression of ERO1L has been reported to be associated with poor prognosis in several cancer indications. However, the role of ERO1L in MM remains unclear. Our data indicate the ERO1L expression is increased in MM cells compared to normal controls. Because MM cells are known to secrete high levels of paraprotein resulting in basal ER stress, we investigated the role of ERO1L in tumor growth. Our data indicate that overexpression of ERO1L in U266 MM cells enhanced cell growth using both in vitro and in vivo models. Moreover, inducible reduction of ERO1L using shRNA strategies inhibited colony formation in U266 cells. Using a pharmacological inhibitor of, ERO1L referred to as EN460, we show that inhibition of ERO1L induced markers of ER stress and triggered apoptosis. Together our findings indicate that ERO1L maybe a tractable strategy for the treatment of MM.

Keywords: multiple myeloma (MM), endoplasmic reticulum oxidase 1 alpha (ERO1L)

Citation Format: Weichih Chen, Werner Geldenhuys, Lori Hazlehurst. Validation of ero1l as a therapeutic target for the treatment of multiple myeloma [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 1915.
Abstract 1922: Targeted silencing of SOX2 by an artificial transcription factor showed antitumor effect in lung and esophageal squamous cell carcinoma
Authors: Yokota Etsuko , Yamatsuji Tomoki , Takaoka Munenori , Haisa Minoru , Takigawa Nagio , Miyake Noriko , Ikeda Tomoko , Mori Tomoaki , Serika Ohno , Sera Takashi , Fukazawa Takuya , Naomoto Yoshio
Published: August 2018

S0X2 is a transcription factor essential for early mammalian development and for the maintenance of stem cells. Recently, SOX2 was identified as a lineage specific oncogene, recurrently amplified and activated in lung and esophageal squamous cell carcinoma (SCC). In this study, we have developed a zinc finger-based artificial transcription factor (ATF) to selectively suppress SOX2 expression in cancer cells and termed the system ATF/SOX2. We engineered the ATF using six zinc finger arrays designed to target a 19 bp site in the SOX2 distal promoter and a KOX transcriptional repressor domain. A recombinant adenoviral vector Ad- ATF/SOX2 that expresses ATF/SOX2 suppressed SOX2 at the mRNA and protein levels in lung and esophageal SCC cells expressing SOX2. In these kinds of cells, Ad-ATF/SOX2 decreased cell proliferation and colony formation more effectively than the recombinant adenoviral vector Ad-shSOX2, which expresses SOX2 short hairpin RNA (shSOX2). Ad-ATF/SOX2 induced the cell cycle inhibitor CDKN1A more strongly than Ad-shSOX2. Importantly, the ATF did not suppress the cell viability of normal human cells. Moreover, Ad-ATF/SOX2 effectively inhibited tumor growth in a lung SCC xenograft mouse model. These results indicate that ATF/SOX2 would lead to the development of an effective molecular-targeted therapy for lung and esophageal SCC.

Citation Format: Etsuko Yokota, Tomoki Yamatsuji, Munenori Takaoka, Minoru Haisa, Nagio Takigawa, Noriko Miyake, Tomoko Ikeda, Tomoaki Mori, Ohno Serika, Takashi Sera, Takuya Fukazawa, Yoshio Naomoto. Targeted silencing of SOX2 by an artificial transcription factor showed antitumor effect in lung and esophageal squamous cell 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 1922.
Abstract 1941: Constitutive IRAK4 activation contributes to NF-κB activity and chemoresistance in colorectal cancer
Authors: Li Qiong , Li Lin , Zhang Daoxiang , Jiang Hongmei , Wang-Gillam Andrea , Ruzinova Marianna B. , Lim Kian-Huat
Published: August 2018

Aberrant activation of the NF-κB transcription factors is a major molecular mechanism that contributes to the aggressive behavior and treatment resistance of colorectal cancer (CRC). Understanding how these factors are activated will provide novel therapeutic opportunities to improve treatment response and outcome of CRC patients. In the present study, we showed that the Interleukin-1 Receptor-Associated Kinases 1 and 4 (IRAK1 and IRAK4) are constitutively activated by phosphorylation in a majority of CRC cell lines and patients samples. Notably, we observe significant positive correlation between phospho-IRAK4 and phospho-NF-kb/p65 staining by immunohistochemistry in patient CRC samples. Moreover, we showed that pharmacologic IRAK4 inhibitors dose-dependently suppressed phosphorylated -IRAK1, -IRAK4 and -IKKa/b by western blots, as well as significantly reducing NF-κB-driven luciferase activity in CRC cells. Furthermore, IRAK4 inhibitor dose dependently inhibit colony formation and anchorage-independent (AI) growth of CRC cells in soft agar. In three CRC cell lines, we observed various degrees of synergism between IRAK4 inhibitors and each chemotherapeutic agents including 5-FU, oxaliplatin and irinotecan, mainly through induction of apoptosis. Lastly, we showed that IRAK4 inhibitor augments the therapeutic effect of 5-FU and oxaliplatin in nude mice bearing CRC xenografts. Together, our data established IRAK4 kinase inhibitors as a promising novel class of targeted agent in CRC.

Citation Format: Qiong Li, Lin Li, Daoxiang Zhang, Hongmei Jiang, Andrea Wang-Gillam, Marianna B. Ruzinova, Kian-Huat Lim. Constitutive IRAK4 activation contributes to NF-κB activity and chemoresistance in colorectal 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 1941.
Abstract 1954: Functional analyses of CLDN18-ARHGAP26 fusion gene in gastric cancers
Authors: Nakayama Izuma , Shinozaki Eiji , Mashima Tetsuo , Kawaguchi Tokuichi , Sakata Seiji , Yamamoto Noriko , Baba Satoko , Dobashi Akito , Togashi Yuki , Sugawara Minoru , Takahashi Shunji , Yamaguchi Kensei , Takeuchi Kengo , Noda Tetsuo
Published: August 2018

[Introduction]Comprehensive molecular analyses by TCGA have classified Gastric Cancers (GC) into 4 groups: EB virus -associated (EBV), Microsatellite instability (MSI), Genomically stable (GS) and Chromosomal instability (CIN). Among these 4 subtypes, patients with GS subtype have few actionable genetic alterations and thus a limited number of molecular-targeted treatments are available. A fusion gene between CLDN18 and ARHGAP26 was first reported by TCGA to be enriched in GS subtype (13.8%), but the prospective functions of this gene product remain unknown.

[Methods]We have previously established 124 GC-Patient derived xenografts (PDXs) from surgically resected specimens in our institute. There are various types of GCs in the PDX panel, including intestinal and diffuse type tumors or tumors of primary and metastatic site. In an attempt to assess roles of CLDN18-ARHGAP26 fusion gene in gastric cancers, we used the cultured cells obtained from GC-PDXs. We first prepared tissue micro-array of formalin-fixed and paraffin-embedded (FFPE) specimen of PDXs and screened for the patients harboring a CLDN18-ARHGAP26 fusion genes by FISH and RT-PCR. In addition, fusion positive cases were sequenced to determine the breakpoints by direct sequencing. Then, we conducted several functional analyses, including assays for cellular proliferation and mobility, by RNAi-knockdown experiments.

[Results]Among 124 GC-PDXs, we detected two CLDN18-ARHGAP26 fusion positive cases. Both of these 2 cases were derived from tumors with diffuse-type GC patient and had identical breakpoint between CLDN18 (exon 5) and ARHGAP26 (exon 10). We successfully established cell lines of these 2 cases. Furthermore, we would verify that PDX derived cells preserved the original patient properties, as transplantation of the PDX-derived cells histologically recapitulated the original tumors. To address its function, we introduced siRNA into cultured cells to reduce their protein levels. Our data so far indicate that the CLDN18-ARHGAP26 fusion might promote anchorage independent cell growth.

[Conclusion] The CLDN18-ARHGAP26 fusion gene potentially contributes to malignant phenotypes of GC. Further analyses in vivo are awaited.

Citation Format: Izuma Nakayama, Eiji Shinozaki, Tetsuo Mashima, Tokuichi Kawaguchi, Seiji Sakata, Noriko Yamamoto, Satoko Baba, Akito Dobashi, Yuki Togashi, Minoru Sugawara, Shunji Takahashi, Kensei Yamaguchi, Kengo Takeuchi, Tetsuo Noda. Functional analyses of CLDN18-ARHGAP26 fusion gene in gastric 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 1954.
Abstract 1976: Identification of 4 novel markers in the chemotherapy resistance of esophageal adenocarcinoma
Authors: Fleegel Jonathan P. , Abdo Joeseph , Agrawal Devendra
Published: August 2018

Esophageal adenocarcinoma (EAC) has seen a steady and steep rise in prevalence since the 1970's. This increase in incidence is correlated with the increase in gastro-esophageal reflux disease and Barrett's esophagus (BE). Unfortunately, where many other cancers have seen progress with advancements in treatments, EAC has not. This cancer is very resistant to almost all first line chemotherapy treatments including the most common platinum based treatments. Although few targeted therapies have been developed, they only show a modest survival advantage for eligible individuals and are not commonly integrated into patient treatment regimens. In our study, we have identified several markers associated with resistance to platinum based chemotherapies that have not previously been documented in EAC. Our study utilized a large-scale discovery mass spectrometric experiment such that the proteome of the 3 different tissue samples could be analyzed, (EAC, BE, and Normal). Part of the novelty of our experiment was that all the tissues collected were from esophagetomies of patients before they had received any chemotherapy. From the data generated we have identified 4 novel markers that are overexpressed in the BE and EAC tissue compared to the normal tissue. In BE tissue DAD1 was over expressed in 9/10 samples, ISG15 (10/10), S100P (10/10) and UBE2N (10/10). In the EAC samples, DAD1 was significantly overexpressed in 20 /20 samples, ISG15 (20/20), S100P (20/20) and UBE2N (19/20). DAD1 inhibits apoptosis and is upregulated by cisplatin and then contributes to cisplatin resistance. S100P has been shown to increase drug resistance in gastric cancer and decrease chemosensitivity 5-FU in vitro in colorectal cancer cells while facilitating proliferation and inhibiting apoptosis. UBE2N plays a role in glycolytic control and in various cellular damage responses. And ISG15 is important in development and metastatic progression of adenocarcinomas through the enhancement of cancer stem cells. We are confident that expression of these four markers can provide clinical utility in assessment of at risk patients with Barrett's esophagus while simultaneously informing treatment regimens. Our goal is to investigate the specific mechanisms by which these markers contribute to cancerous development and chemotherapy resistance in order to elucidate targets for future treatment of EAC.

Citation Format: Jonathan P. Fleegel, Joeseph Abdo, Devendra Agrawal. Identification of 4 novel markers in the chemotherapy resistance of esophageal adenocarcinoma [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 1976.
Abstract 2003: P38α suppresses tumor initiating cell phenotypes through inhibition of NFATc4 in non-small cell lung cancer
Authors: Xiao Zhi-Jie , Liu Jing , Wang Si-Qi , Gao Xu-Yuan , Tin Vicky Pui-Chi , Wong Maria Pik
Published: August 2018

Lung cancer development is often mediated through oxidative and inflammatory stresses caused by tobacco and environmental carcinogens, suggesting dysregulation of stress mediators might play a role during carcinogenesis. The p38 MAPK pathway is activated by multiple stress stimuli and inflammatory cytokines, and mediates a wide range of cellular processes in a context-specific manner. P38α, a member of the p38 family, is known to regulate the self-renewal and differentiation of adult lung stem cells. While p38α has been reported to mediate tumor suppressive or supportive roles in different cancers, inactivation of p38α in the lung epithelium is involved in KRASG12V induced tumorigenesis. However, whether p38α could be involved in regulating tumor initiating cells (TIC) of lung cancer remains unclear. To investigate this possibility, immunohistochemistry was first used to analyse the nuclear expression of phosphorylation-activated p38α (p-p38α) in 90 human primary lung adenocarcinomas. Results showed higher expression of p-p38α was significantly correlated with longer progression free and overall survivals, as well as a more differentiated tumor histological phenotype, indicating p38α might play a tumor suppressive role and drive tumor differentiation in lung cancer. To further address its functional role on lung TIC, p38α was suppressed by siRNA or pharmacologically inhibited by SB203580. This led to increase of the TIC subset that coexpresses ALDH and CD44 (ALDH+/CD44+-TIC), as well as up-regulated expression of the pluripotency genes SOX2, OCT4, and NANOG in lung cancer cell lines. Functionally, p38α inhibition by SB203580 promoted tumor spheres formation for two consecutive generations. NFAT is a family of transcription factors reported to regulate TIC in various cancers which are activated via de-phosphorylation leading to nuclear translocation. We hypothesize the TIC supportive function of p38α inhibition might be mediated through loss of phosphorylation and subsequent activation of NFATc4. In line with this hypothesis, we showed, using Western blot, p38α inhibition increased the expression of nuclear NFATc4. Moreover, NFATc4 knockdown led to suppressed sphere formation and pluripotency genes expression in lung cancer cell lines. Together, our results indicated p38α mediates a tumor suppressor pathway through downstream NFATc4 inactivation and TIC inhibition. The p38α -NFATc4 stress pathway might serve as a potential target for long term lung cancer treatment through TIC regulation.

Citation Format: Zhi-Jie Xiao, Jing Liu, Si-Qi Wang, Xu-Yuan Gao, Vicky Pui-Chi Tin, Maria Pik Wong. P38α suppresses tumor initiating cell phenotypes through inhibition of NFATc4 in non-small cell lung 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 2003.
Abstract 2008: Stem cell-associated proteins P63 and Bmi-1 may cooperate in maintaining cancer-initiating cell populations in squamous cell carcinomas
Authors: Ha Linan , Reilly Devin , Ponnamperuma Roshini , George Andrea , Weinberg Wendy C.
Published: August 2018

Overexpression of p53-related protein p63, particularly ΔNp63α, is seen in squamous cell carcinomas of the head and neck, lung, cervix and skin. We use primary murine epidermal keratinocytes as a model of human squamous epithelial cells and mimic these elevated levels using adeno- and lentiviral vectors. In previous studies, we established that ΔNp63α inhibits induction of p16ink4a and p19arf, blocks oncogene-induced senescence, and cooperates with activated v-rasHa to enhance conversion of papilloma to carcinoma in a nude mouse grafting model. We evaluated the relationship between ΔNp63α and Bmi-1, a member of the polycomb complex known to repress the INK4a/ARF gene locus, and modulated Bmi-1 expression in the presence or absence of elevated ΔNp63α to assess its impact on p16ink4a and p19arf expression. Bmi-1 protein levels correlate with those of ΔNp63α; however, higher levels of Bmi-1 block p16ink4a but not p19arf. Consistent with this, Bmi-1 is required for the binding of ΔNp63α to the p16ink4a but not the p19arf promoter. These findings may have further important implications in HNSCC as other previously published models have demonstrated that Bmi-1 is important for prevention of senescence in stem cells. Using FACS analysis and cell sorting, we are defining cell populations of keratinocytes with respect to the expression of ΔNp63α, Bmi-1, and other markers of stem cells and senescence and extending these approaches from murine to human HNSCC models with the aim of correlating these distinct patterns with cancer-initiating potential and tumor phenotype.

Citation Format: Linan Ha, Devin Reilly, Roshini Ponnamperuma, Andrea George, Wendy C. Weinberg. Stem cell-associated proteins P63 and Bmi-1 may cooperate in maintaining cancer-initiating cell populations in squamous cell carcinomas [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 2008.
Abstract 2023: RhebL1 is involved in epithelial mesenchymal transition of breast cancer cells
Authors: KIM Hyunji , LEE Changhoon
Published: August 2018

RhebL1 belongs to the ras superfamily of G proteins and is highly conserved across many species. However, the cellular function of RhebL1 have not yet been reported especially in breast cancer. In this study, we found that RhebL1 is highly expressed in MDA-MB-231 and SK-BR-3 breast cancer cells. By using the public microarray data sets, we found that overall and relapse free survival were decreased in breast cancer patients with high expression of RhebL1. The involvement of RhebL1 in epithelial mesenchymal transition of breast cancer cells was examined. Gene silencing of RhebL1 decreased the expression of mesenchymal markers such as N-cadherin, Vimentin, snail and slug and increased the expression of epithelial marker E-cadherin in MDA-MB-231 and SK-BR-3 breast cancer cells. In contrast, overexpression of RhebL1 increased the expression of mesenchymal markers and decreased the expression of E-cadherin in MCF7 breast cancer cells. Next, we examined the effects of RhebL1 expression on migration and invasion since migratory and invasive capabilities were strongly related to EMT. RhebL1 overexpression significantly increased the migration and invasion of MCF7 cells and gene silencing of RhebL1 reduced the migration and invasion of MDA-MB-231 and SK-BR-3 cells. Furthermore, we identify a novel RhebL1-binding protein, RBP1, which mediates the segregation of protein phosphatase 2A from Akt, leading to induced Akt phosphorylation under conditions by binding to RhebL1. The collective results indicate that RhebL1 is involved in EMT of breast cancer cells via interaction with RBP1-Akt-PP2A complex. These results suggest that RhebL1 may be a key regulator modulating metastatic breast cancer cells.

Citation Format: Hyunji KIM, Changhoon LEE. RhebL1 is involved in epithelial mesenchymal transition of breast 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 2023.
Abstract 2031: The noncanonical pathways of transforming growth factor β in ovarian carcinoma
Authors: Catane Liora Jacobs , Trope Claes G. , Davidson Ben , Reich Reuven
Published: August 2018

Introduction: Of all the gynecological malignancies, ovarian cancer (OC) has the highest mortality rate. This can be attributed to the late detection and acquired drug resistance. It metastasizes mainly in the serosal cavities, and produces cancerous cells dispersed in the peritoneal and pleural fluids called effusions. Transforming Growth Factor β (TGFβ) is a family of growth factors that play a significant role in cellular processes. There are three homologous isoforms of TGFβ expressed in humans: TGFβ1-3. These 3 ligands activate 3 transmembrane receptors, TβRI-III. The 3 receptors and the ligand TGF-β2 have different splice variants. TGFβ signaling induces canonical and non-canonical signaling. The Non-canonical pathways consist of a variety of different pathways, including MAP kinase, Rho-like GTPase, and phosphatidylinositol-3-kinase (PI3K)/ AKT pathways.

Aims and methods: Our first aim was to investigate the metastatic role of TGFβ receptor variants. This was accomplished through the creation of specific receptor variant knockouts (KO's) using the CRISPR/Cas9 genome editing system and subsequent metastatic tests. Next we characterized chosen non-canonical pathways and downstream effectors in the CRISPR KO's through Western Blot analysis and qPCR respectively. The Second aim of this study was to characterize the non-canonical pathways In-vivo. Using human tumor tissue samples, we examined the expression and activation of the different pathways via Western Blot analysis and checked for correlation with clinical parameters.

Results: KO cells showed significantly lower migration and invasion capabilities compared to Wild Type (WT) cells. Our results show a decrease in the activation of ERK1/2, JNK1 and JNK2 in the TβRI variant 1 KO in ES-2 cells (p=0.058, p=0.002 and p<0.0001 respectively). The downstream effectors SNAIL and TWIST show significantly higher levels in the KO compared to WT cells. Of the pathways examined, p-AKT was found to be higher in post chemotherapy samples (p=0.029) and JNK1 was higher in effusions from patients with poor chemotherapy response (p=0.045). Site-related findings show that p85 (PI3K), AKT and p-AKT were highest in metastasis, medium in solid ovarian carcinoma and lowest in effusions (p<0.0001, p=0.0024 and p=0.0021 respectively).

Closing statement: These findings provide new insight on the importance of TGFβ‘s role in the progression of OC, and is, as far as we know, is the first study done on the metastatic roles of the receptor variants.

Citation Format: Liora Jacobs Catane, Claes G. Trope, Ben Davidson, Reuven Reich. The noncanonical pathways of transforming growth factor β in ovarian 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 2031.
Abstract 2032: CXCR7 promotes the migration and invasion of head and neck squamous cell carcinoma through Smad2/Akt signaling
Authors: Kim Nayoung , Kim Solbi , Ryu Hyewon , Lee Hyo Jin
Published: August 2018

Background: The chemokine receptor, CXCR7, has been shown to play an important role in the progression of several types of cancer. However, there have been few reports on the biological role of CXCR7 in head and neck squamous cell carcinoma (HNSCC). In this study, we investigated the functional role of CXCR7 and the underlying molecular mechanism of disease progression in HNSCC.

Methods: We characterized the expression of CXCR7 in tumor specimens from patients with HNSCC. The biological functions of CXCR7- and CXCR7-mediated signaling pathways were investigated in HNSCC cell lines with CXCR7 overexpression and treatment of SDF-1α, a major ligand of CXCR7, as well as knockdown of CXCR7.

Results: CXCR7 was differentially expressed in human HNSCC patients. High expression of CXCR7 was associated with an aggressive tumor behavior. Overexpression of CXCR7 dramatically promoted cell migration and invasion in HNSCC cells, although it was not involved in cell proliferation and colony formation. CXCR7 knockdown using siRNA in HNSCC cells recovered the cell migratory and invasive behavior of HNSCC cells. CXCR7 overexpression also induced the epithelial-mesenchymal transition. Vimentin, Slug, and Twist were increased but E-cadherin and Ep-CAM were decreased by CXCR7 expression. Akt phosphorylation and Smad2 signaling activation were induced in HNSCC cells with CXCR7 overexpression. Treatment with a PI3K inhibitor reduced Slug and Twist levels while suppression of Smad2 signaling by siRNA reduced Akt phosphorylation, as well as Slug and Twist. Furthermore, inhibition of Smad2 decreased tumor cell migration and invasion in HNSCC.

Conclusions: CXCR7 contributed to cell migration and invasion of HNSCC through the Smad2/Akt signaling axis, suggesting that CXCR7 might be a therapeutic target for the treatment of HNSCC.

Citation Format: Nayoung Kim, Solbi Kim, Hyewon Ryu, Hyo Jin Lee. CXCR7 promotes the migration and invasion of head and neck squamous cell carcinoma through Smad2/Akt signaling [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 2032.
Abstract 2040: Withaferin A inhibits epithelial-to-mesenchymal transition in non-small lung cell cancer cells via regulation of SMAD and NFkB signaling
Authors: Kyakulaga Al Hassan , Aqil Farrukh , Munagala Radha , Gupta Ramesh
Published: August 2018

Background: Metastasis remains the primary cause in lung cancer. Epithelial-to-mesenchyme transition (EMT) has been identified as the hallmark for metastasis in NSCLC, and is being explored for drug targeting. Aberrant signaling mechanisms for cytokines such as TGF-β1 and TNFα are known to induce EMT. In this study we demonstrate the effect and mechanisms of WFA inhibition of EMT in NSCLC cell lines.

Objective: The present study was done to determine the effect of WFA on TGF-β1 and TNFα induced EMT, migration and invasion in human NSCLC cells. Furthermore, to determine underlying the mechanisms of WFA inhibition of EMT.

Methods: Two human NSCLC cell lines (H1299 and A549) were cultured in DMEM media. To induce the EMT, cells in serum free media were challenged with TGF-β1 (5 ng/mL) or TNFα (5 ng/mL) alone or in combination. To determine the effects of WFA, cells were either pre- or co-treated with different concentrations of WFA for various time points. Western blot analysis, electrophoretic mobility shift assay (EMSA), RT-PCR and immunofluorescence staining were used to determine effects of WFA on Smad2/3 and NFkB activation. Cell adhesion, migration and invasion assays were used to assess in vitro anti-metastatic activity.

Results: Our findings indicate that the cytokines TGF-β1 and TNFα cooperate to promote the acquisition of EMT phenotype, migration and invasion of NSCLC cells in vitro. There was a marked increase in the expression of EMT transcription factors Snail, Slug, ZEB and EMT markers such as vimentin and N-cadherin. Cells treated with TGF-β1/TNFα had significantly increased migratory and invasive capacities. WFA treatment significantly inhibited the TGF-β1 induced Smad2/3 activation and TNFα-induced translocation of NFkB into the nucleus. We observed a dose and time dependent inhibition of the phosphorylation of Smad2/3 and NFkB in both cell lines. The cell adhesion, migratory and invasive capacities of both H1299 and A549 cells were greatly diminished by WFA in the presence or absence of cytokines. Conclusion: Based on the current finding, it can be concluded that WFA inhibits EMT via negative regulation of Smad2/3 and NFkB signaling in NSCLC cells.

Citation Format: Al Hassan Kyakulaga, Farrukh Aqil, Radha Munagala, Ramesh Gupta. Withaferin A inhibits epithelial-to-mesenchymal transition in non-small lung cell cancer cells via regulation of SMAD and NFkB signaling [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 2040.
Abstract 2054: Tumor endothelial cells survive under lactic acidosis
Authors: Annan Dorcas A. , Maishi Nako , Soga Tomoyoshi , Dawood Randa , Hida Yasuhiro , Hida Kyoko
Published: August 2018

Tumor endothelial cells (TECs) which line tumor blood vessels differ functionally, structurally and genetically from normal endothelial cells (NECs). Recent findings have furthermore shown that TECs are hyper-glycolytic, making them a significant source of lactic acid within the tumor. Similar to TECs, tumor cells are also preferably glycolytic and release large amounts of lactic acid. Consequently, TECs may be influenced by both intrinsic and tumor-derived lactic acid. However, the effects of lactic acidic on TECs have not been elucidated. Moreover, with the quest to find novel therapeutic antiangiogenic targets, it is of much essence to identify adaptive mechanisms supporting TECs under conditions of lactic acidosis. In this study, metabolomic analysis was performed in TECs and NECs. TECs had significantly higher intracellular levels of glycolytic metabolites, lactate, ATP and amino acids than NECs. Additionally, unlike NECs, TECs proliferated when exposed to conditions of lactic acidosis (20mM lactate, pH 6.9) and not under lactosis (20mM lactate, pH 7.3). This observation implied that TECs survive and sustain their growth under low pH conditions but in the presence of lactate. Furthermore, qRT -PCR analysis revealed an elevated expression of proton-coupled transporters in TECs than in NECs. The TECs were sensitive to drugs targeting some of the transporters. This indicated that proton-coupled transporters may be involved in the survival and proliferation of TECs in lactic acidosis. Next, the effects of lactic acidosis on tumor blood vessels, particularly endothelial cellular junctions was investigated. VE-cadherin expression was analysed by immunocytochemistry in endothelial cells exposed to lactosis and lactic acidosis. The VE-cadherin was more disorganized under lactic acidosis than in lactosis. Lactic acidosis may therefore contribute to the poor integrity of tumor blood vessels. More studies are on-going to elucidate the molecular effects of lactic acidosis on TECs and the role played by proton-coupled transporters in TECs survival in the tumor microenvironment. These will lead to the identification of a proton-coupled transporter as an antiangiogenic therapeutic target.

Citation Format: Dorcas A. Annan, Nako Maishi, Tomoyoshi Soga, Randa Dawood, Yasuhiro Hida, Kyoko Hida. Tumor endothelial cells survive under lactic acidosis [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 2054.
Abstract 2070: The prevalence of DICER1 pathogenic variation in cancer population databases
Authors: Kim Jung , Schultz Kris Ann P. , Hill D. Ashley A. , Stewart Douglas R.
Published: August 2018

DICER1 is an RNaseIII endonuclease that is crucial for processing pre-microRNA into mature microRNA. The DICER1 syndrome is a rare, autosomal dominant tumor-predisposition disorder associated with pleuropulmonary blastoma, a rare pediatric lung cancer. Somatic missense variation in “hotspot” codons in the RNaseIIIb domain (E1705, D1709, G1809, D1810, E1813) is observed in DICER1-associated tumors. Previously, we found in the general population (non-TCGA ExAC) that the prevalence of pathogenic DICER1 variation is approximately 1:10,000. In this study, we investigated the prevalence of pathogenic DICER1 variation in common adult and pediatric cancers. We examined two publicly-available cancer datasets: TCGA (adult; 32 cancer types; 9,173 exomes) and TARGET (pediatric; 3 cancer types; 236 exomes). TCGA bam files were downloaded and germline variants were called. Germline VCF files were obtained from TARGET investigators. All datasets were annotated with snpEff and ANNOVAR. Variants were binned into 4 categories: likely benign (LB), variant of unknown significance (VUS), likely pathogenic (LP), or pathogenic (P). We reviewed publicly-available pathology images for the TCGA tumors. The prevalence of DICER1 P/LP variants (in all cancers combined) was 1:834 and 1:118 in TCGA and TARGET, respectively. To reduce potential prevalence inflation, we conducted a more stringent prevalence calculation by only considering loss-of-function and hotspot variants. The prevalence of P variants was approximately 1:4586 in TCGA and 1:236 in TARGET. With these more stringent criteria, a single participant with a uterine corpus endometrial carcinoma (UCEC in TCGA) harbored a two pathogenic germline DICER1 (hotspot and splice donor) variants, and a second UCEC harbored a somatic DICER1 (hotspot) variant. In TARGET, a single participant with a neuroblastoma harbored a germline DICER1 canonical splice variant. There were no atypical or unusual features noted on pathology review of publicly-available slides from TCGA tumors. In TCGA, across many types of cancer, the prevalence of pathogenic DICER1 variation was higher compared with the general population (~1:4,600 vs. ~1:10,000), a finding driven by one cancer type (UCEC). In the smaller TARGET dataset, we observed a single pathogenic germline variant in neuroblastoma. This is the first comprehensive analysis of DICER1 pathogenic variation in large adult and pediatric cancer populations. The novel association of DICER1 variation with UCEC merits additional investigation.

Citation Format: Jung Kim, Kris Ann P. Schultz, D. Ashley A. Hill, Douglas R. Stewart. The prevalence of DICER1 pathogenic variation in cancer population databases [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 2070.
Abstract 2085: Exploring mutation signatures in pediatric cancers
Authors: Lucas Francis A. San , Lupo Philip , Brown Austin , Scheurer Michael , Scheet Paul
Published: August 2018

Background: Mutation signatures have been catalogued across most common cancers, providing insights into their underlying mutational processes and suggesting strategies for categorization and subphenotyping. However, these analyses have been limited to mostly adult cancers, likely due to the relative rarity of pediatric analogs and a corresponding lower mutation burden of these tumors. Here we attempted to compare mutation signatures across several pediatric cancers, given these constraints, with their corresponding adult versions.

Data and Methods: From the ICGC data portal, we downloaded clinical and mutation data for pediatric brain (medulloblastoma, glioblastoma, neuroblastoma) and blood-based (acute lymphoblastic leukemia, acute myeloid leukemia) cancers. We also obtained data for related adult cancers, including glioblastoma and acute myeloid leukemia. For each cancer, we characterized mutation patterns using trinucleotides centered at somatic SNVs and then estimated the composition of underlying putative COSMIC mutation signatures. For cancers with low mutation rates (impeding estimates of mutation signatures), we grouped mutations from similar samples based on clinical and molecular statuses to infer mutation signatures.

Results: Each of the pediatric cancer types strongly exhibited the common cancer mutation Signature 1 (spontaneous deamination of 5-methylcytosine), as might be expected since it has been described across all adult cancer types. Further, pediatric acute myeloid leukemia and glioblastoma both harbored mutation signatures seen in their adult counterparts in addition to unexpected patterns of defective DNA mismatch repair and DNA double-strand break repair. Interestingly, for pediatric glioblastoma we identified Signatures 3, 8, 15 and 16, which were previously unidentified in adult glioblastoma and have been recently implicated in pediatric medulloblastoma.

Conclusion: Our findings may be influenced by the sparsity of mutations in pediatric cancers, highlighting the need for quantifying precision in measures of mutation signatures. This notwithstanding, from our initial observations, pediatric cancers appear to exhibit a more diverse set of mutation signatures than their adult counterparts. Further, pediatric cancers of different histologies, particularly those of brain cancers, may share molecular mechanisms more than currently believed.

Citation Format: Francis A. San Lucas, Philip Lupo, Austin Brown, Michael Scheurer, Paul Scheet. Exploring mutation signatures in pediatric 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 2085.
Abstract 2094: Pathology beyond 2D: Imaging and analysis of cells and the tumor microenvironment in three dimensions
Authors: Mansfield James R. , Wainright James , Bagnall Christopher , Wilde Geraint , Price Meredith , Willcox Benjamin E. , Browne Mark
Published: August 2018

The success of cancer immunotherapy treatments and the development of immuno-oncology into the “fifth pillar” of cancer treatment has fueled a rapid growth in interest into methodologies that investigate immune cells in the context of the tumor microenvironment. However, nearly all technologies that investigate the immune contexture of tumors either rely on homogenization or disaggregation of the sample and the concomitant loss of spatial relationships, or rely on a thin section (5 um or less), two-dimensional sample of a biopsy, which is a poor representation of an inherently three-dimensional structure. An image of a thin section will tend to over-estimate the numbers of proximal cells and underestimate the numbers of more distant cells due to sampling error. To address this, we have implemented an imaging system capable of 3D confocal and super-resolution imaging, tiling of large sample areas and imaging samples over time along with software for the visualization and analysis of the datasets.

We present here a series of examples showing the utility of 3D fluorescence imaging of labeled cells in thick (ca. 50 micron) FFPE tissue sections. By using a multimarker immunostaining scheme, this enables the phenotyping of cells within the tumor microenvironment and the calculation of distance metrics between cells in three dimensions. This methodology is an inherently better means of assessing cellular distributions in the tumor microenvironment and can give a much better representation of the complicated 3D structure of a tumor than imaging of thin sections.

Citation Format: James R. Mansfield, James Wainright, Christopher Bagnall, Geraint Wilde, Meredith Price, Benjamin E. Willcox, Mark Browne. Pathology beyond 2D: Imaging and analysis of cells and the tumor microenvironment in three dimensions [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 2094.
Abstract 2101: The growth of a xenograft breast cancer tumor model with engineered hyaluronan-accumulating stroma is dependent on hyaluronan and independent of CD44
Authors: Zhao Chunmei , Thompson Benjamin J. , Chen Kelly , Marella Mathieu , Zimmerman Susan , Kimbler Trevor , Blouw Barbara , Garrovillo Sheryl , Huang Lei , Radi Adrian , Huang Zhongdong , Shepard H. Michael , Rosengren Sanna , Thanos Christopher D. , Maneval Daniel C.
Published: August 2018

The abnormal accumulation of hyaluronan in the solid tumor microenvironment is associated with poor prognosis in several human cancers. Hyaluronan often accumulates in the tumor stroma. To understand the role of hyaluronan-accumulating stroma in tumor progression, we engineered a hyaluronan-producing fibroblast cell line 3T3HAS3 by lentiviral transduction of the human hyaluronan synthase 3 gene into Balb/c 3T3 cells. The 3T3HAS3 cells produced significant amounts of hyaluronan in vitro, which bind to the cell surface of the human breast cancer cell line MDA-MB-468. When co-grafted with MDA-MB-468 cells in nude mice, 3T3HAS3 significantly enhanced tumor growth. Immunohistochemical analysis of tumor xenografts showed that MDA-MB-468 cells were surrounded by hyaluronan-accumulating stroma, closely resembling the morphology observed in human breast cancer specimens. Tumor growth of this co-graft model required hyaluronan production from 3T3HAS3 cells, as demonstrated by the delayed tumor growth upon hyaluronan removal by expression of the human PH20 gene in 3T3HAS3 cells, or by systemic administration of PEGylated recombinant human PH20 (PEGPH20). In contrast, neither the expression of CD44, a well-characterized hyaluronan receptor, in tumor and stromal fibroblasts, nor the binding of hyaluronan to CD44 in MDA-MB-468 tumor cells was essential for tumor growth. Small scale screening of signaling changes in xenograft tumors suggested that the AMPK/mTOR pathway may respond to hyaluronan removal by PEGPH20. Collectively, these data demonstrate that the growth of an engineered breast cancer xenograft model with hyaluronan-accumulating stroma is dependent on hyaluronan, and that hyaluronan-CD44 interaction may not be the main mechanism through which hyaluronan promotes tumor progression in certain tumors.

Citation Format: Chunmei Zhao, Benjamin J. Thompson, Kelly Chen, Mathieu Marella, Susan Zimmerman, Trevor Kimbler, Barbara Blouw, Sheryl Garrovillo, Lei Huang, Adrian Radi, Zhongdong Huang, H. Michael Shepard, Sanna Rosengren, Christopher D. Thanos, Daniel C. Maneval. The growth of a xenograft breast cancer tumor model with engineered hyaluronan-accumulating stroma is dependent on hyaluronan and independent of 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 2101.