bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 540: β-Catenin/LIN28B promotes cell proliferation of human trophoblast cells via let7a repression
Authors: Wu Jing , Luan Baoxin , Yu Huandi , Yu Yinhua , Xu Congjian , Zhao Hongbo
Published: August 2018

LIN28 is a highly conserved RNA-binding proteins, have been implicated in key biological functions such as development, glucose metabolism and pluripotency. LIN28 overexpression induces the reprogramming of pluripotent stem cells (iPSCs) and supports the proliferative and metabolic capacities of iPSCs. Therefore LIN28 is crucial for the regulation of the cell fate and human embryonic development. In addition, LIN28B is frequently up-regulated in a variety of cancers and correlates with poor prognosis and cancer recurrence, indicating the crucial role of LIN28B in tumorigenesis. Aberrantly expression of LIN28B is responsible for the decrease in the let-7 microRNA family observed in many cancers and Let-7 is frequently down-regulated in numerous different tumors and lower levels of let-7 correlates with poor prognosis. Therefore, LIN28/Let7 may be crucial for the regulation of cell cycle and cell proliferation. In our present study, We first detected the expression of LIN28B in normal placental villi and choriocarcinoma tissue. Our data showed LIN28B was highly expressed in human choriocarcinoma tissues and most LIN28B staining was localized in the nucleus. Comparatively, LIN28B staining was faint in normal placental villi. Our data then demonstrated that LIN28B was highly expressed in human choriocarcinoma cell lines JEG-3 and JAR. However, LIN28B expression was undetectable in HTR-8/Svneo normal trophoblast cells. Our data then demonstrated that LIN28B knockdown by small interfering RNA (siRNA) caused an increase in Let-7a expression. Further, silencing of LIN28 inhibited IGF2BP1 expression and suppressed cell proliferation capacity, which can be markedly restored by Le7a siRNA. These findings reveal that LIN28 may promote IGF2BP1 expression and cell proliferation by Let-7a repression. Moreover, β-catenin knockdown resulted in reduced LIN28 expression and Let-7a up-regulation. β-catenin is the major mediator of canonical Wnt signaling pathway, and activation of canonical Wnt signaling has been reported to play a critical role in the proliferation, invasion, and differentiation of various tumors. Collectively, our present data indicated that β-catenin/LIN28B/Let7a pathway may be crucial for cell proliferation regulation in human choriocarcinoma cells.

Citation Format: Jing Wu, Baoxin Luan, Huandi Yu, Yinhua Yu, Congjian Xu, Hongbo Zhao. β-Catenin/LIN28B promotes cell proliferation of human trophoblast cells via let7a repression [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 540.
Abstract 541: Involvement of miRNA expression may contribute for FoxO3a oncogenic role in uterine leiomyosarcoma
Authors: Garcia Natalia , Ricci Anamaria Ritti , Almeida Bruna Cristine de , Almeida Thais Gomes , Cunha Isabela Werneck , Baracat Edmund Chada , Carvalho Kátia Candido
Published: August 2018

Introduction: Leiomyoma (LM) and leiomyosarcoma (LMS) are uterine mesenchymal tumors that present variable clinical behavior. LM are extremely common estrogen and progesterone dependent benign tumors. On the other hand, LMS are rare and aggressive malignant tumors with limited available treatment options. FoxO3a seems to have a tumor suppressor role and its loss of function may determine deregulation in the cell proliferation, with DNA damage accumulation. Several studies demonstrated FoxO3a with an important role in several tumors development; however, its regulation or function is unknown in uterine mesenchymal tumors. Objective: Here, our goals were to assess the FoxO3a expression profile in uterine LM and LMS, to evaluate the mechanisms involved on FoxO3a regulation, and to assess its use as a marker for diagnosis, prognosis and malignancy risk prediction. Methods: Formalin-Fixed Paraffin-Embedded tissues of 56 leiomyosarcomas (LMS), 80 leiomyomas (LM) and 20 myometrium (MM) from patients, were used for FOXO3a protein evaluation by immunohistochemistry (IHQ). Epigenetic regulation of FoxO3a expression was assessed by 84 miRNAs gene analysis, using Real Time PCR method (MIHS 109Z, Qiagen). MM, LM and LMS cells were evaluated for FoxO3a gene expression by Real Time PCR. Results: FOXO3a protein showed an increased expression profile in MM, LM and LMS. Higher protein levels were observed in the comparison between LMS and MM (p<0,05). Gene expression analysis corroborate the protein data, with higher FoxO3a expression in LMS compared to MM and LM cells. Concerning the miRNAs analysis, we found oncogenic has-96-5p and has-155-5p overexpressed in LMS samples (p=0,008 and p=0,0007; respectively), while the tumor suppressor miR-let7c-5p was down expressed (p=0.0026). Conclusion: FoxO3a seems to display an oncogenic role in uterine tumors and could participate in the tumors risk of malignancy. miRNAs expression profile corroborates the FoxO3a negative regulation in these tumors. However, further studies are necessary for the understanding of the FoxO3a role in LM and LMS biology.

Citation Format: Natalia Garcia, Anamaria Ritti Ricci, Bruna Cristine de Almeida, Thais Gomes Almeida, Isabela Werneck Cunha, Edmund Chada Baracat, Kátia Candido Carvalho. Involvement of miRNA expression may contribute for FoxO3a oncogenic role in uterine leiomyosarcoma [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 541.
Abstract 5430: Evaluating a novel phytobiologic mixture against breast cancer cell lines: Effect on glycolytic enzymes and EMT gene expression profile
Authors: Alamoudi Aliaa A. , Alqarni Afnan A. , Ajabnoor Ghada , Niedwiecki Aleksandra , Rath Matthias , Harakeh Steve M. , Al-Abd Ahmed M.
Published: August 2018

Background: Recently, there has been a growing evidence proposed regarding role of phytobiological compounds against various malignancies. One of the novel phytobiological mixture (PB), which is a mixture composed of the following: quercetin, resveratrol, cruciferex, epigallocatechin-3-gallate and curcumin at different proportions. To the best of our knowledge, this is the first study to evaluate the effects of a PB on breast cancer cell lines and its impact on the expression profile of these cells.

Aim: Herein, we investigated the anti-proliferative effects of PB against two breast cancer cell lines: MDA-MB-231 and MCF-7, in addition, to investigating whether PB may alter the expression profile of glycolytic enzymes and epithelial mesenchymal transition (EMT) genes in these cell lines.

Method: The anti-proliferative effect of PB was evaluated in vitro against MCF-7 and MDA-MB-231 cell lines using SRB assay. The effect of PB on the relative expression profile of some of the glycolytic enzymes and EMT genes was conducted using TaqMan gene expression assays via reverse transcription-quantitative PCR (RT-qPCR).

Results: At 72 hrs, treatment with PB showed cytotoxic effects against MDA-MB-231 and MCF-7 cells with an IC50's of 25.9±3 µg/ml and 29.5± 0.9 µg/ml, respectively. At 24 hrs post PB treatment, there was a significant increase in the gene expression of most glycolytic enzymes such as; Phosphofructokinase muscle type (PFKM) (1.4± 0.1, p<0.01) and Pyruvate dehydrogenase kinase isoenzyme 1 (PDK-1) (1.6± 0.2 fold, p< 0.001). PB did not seem to alter the EMT gene expression profile with both cadherin 1 and 2 showing no significant changes. Interestingly, however, a significant up-regulation was seen with ZEB2 member of the zinc finger E-box binding homeobox 2, post PB treatment.

Conclusion: In conclusion PB showed a promising anti-proliferative effect against two breast cancer cell lines. However, we postulate that the drug could be modulating glycolytic enzymes expression, which ultimately can affect key molecules in cell differentiation. Further studies are needed to elucidate the exact underlying mechanism and to better understand the most beneficial drug combinations that can work synergistically with PB to interfere with different tumor hallmarks.

Citation Format: Aliaa A. Alamoudi, Afnan A. Alqarni, Ghada Ajabnoor, Aleksandra Niedwiecki, Matthias Rath, Steve M. Harakeh, Ahmed M. Al-Abd. Evaluating a novel phytobiologic mixture against breast cancer cell lines: Effect on glycolytic enzymes and EMT gene expression profile [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 5430.
Abstract 5446: Serotonin modulates AKT-mTOR and Notch signaling pathways, promotes liver cancer cell steatosis and cell survival
Authors: Niture Suresh , Kumar Deepak
Published: August 2018

The hormone serotonin is known as 5-hydroxytryptamine. It is a precursor of melatonin, derived from the amino acid tryptophan. Serotonin acts as a neurotransmitter and modulates several key functions in the human body, such as appetite, mood and sleep. Apart from its important roles in the central nervous system (CNS), serotonin regulates various extra-neuronal pathophysiological functions. Serotonin is synthesized by Enterochromaffin cells of the gut, mostly stored in platelets and about 2% of free serotonin released in the blood. The role of peripheral serotonin in adipocytes, pancreatic cells, macrophages and liver cells is not fully understood. In the current study, we investigated the role of serotonin in liver cancer cell signaling and liver cancer cell growth. Treatment of serotonin in liver cancer cells HepG2 and SK-Hep1 induced autophagy markers and effectors such as LC3 I/II, phospho-4EBP1, ATG3 and Beclin1, and, increased cell proliferation and cell survival. AKT/mTOR pathway controls cellular autophagy and our results demonstrated that exposure of serotonin to liver cancer cells inactivates AKT/mTOR signaling and induced autophagy. Also, we demonstrate that serotonin also activates Notch signaling pathway as evident by induction of Notch target gene Hes1. Notch inhibitors Avagacestat 2 and FLI-06 inhibited serotonin-mediated Notch signaling and cellular autophagy. Furthermore, by the activation of Notch signaling, serotonin-induced oleic acid-mediated cell steatosis in both HepG2 and SK-Hep1 liver cancer cells. In addition, treatment of serotonin also induced autophagy modulator proteins such as SIRT1 and Tumor Necrosis Factor Alpha Inducing Protein 8 (TNFAIP8) which participate in the modulation of cellular autophagy and cell survival. Taken together our data suggested that peripheral serotonin regulates liver cancer cell steatosis, cells survival and may promote liver carcinogenesis by the modulation of AKT/mTOR and Notch signaling pathways.

Citation Format: Suresh Niture, Deepak Kumar. Serotonin modulates AKT-mTOR and Notch signaling pathways, promotes liver cancer cell steatosis and cell survival [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 5446.
Abstract 5459: Regulation of IGF2 by TGF-β signaling in liver cancers and stem cell homeostasis
Authors: Rao Shuyun , Zaidi Sobia , Ohshiro Kazufumi , Chen Jian , Gu Shoujun , Jogunoori Wilma , White Jon , Mishra Bibhuti , Li Shulin , Akbani Rehan , Mishra Lopa
Published: August 2018

Development of hepatocellular carcinoma (HCC), which remains lethal, is associated with alterations in multiple factors including the transforming growth factor beta (TGF-β) signaling pathway. Previously we have uncovered a unique role for TGF-β signaling molecules, Smad3 and its adaptor β2SP, in suppressing stem cell transformation into cancer. Yet, while TGF-β plays a pleiotropic role including regulating stem cell differentiation, proliferation, and inflammation, mechanistic insight into the dichotomy of TGF-β, and its role in stem cell transformation remains poorly understood for these cancers. Here, we took an integrated approach to identify and validate effects of changes in this pathway in HCC and identify potential therapeutic targets such as IGF2. We extended our mechanistic studies associated with the regulation of insulin-like growth factor 2 (IGF2) in the context of the TGF-β-pathway, by utilizing both human liver cancer cell lines and TGF-β signaling-deficient mice (β2SP+/- and β2SP+/-/Smad3+/-) that develop liver cancers. We found the following: 1) IGF2 promotes stemness, overexpression leading to increased expression of stem cell genes that include SOX2 and ALDH1A1, and sphere formation in liver cancer cells; 2) A marked increase in IGF2 mRNA and protein in HepG2 cells and Huh7 cells with β2SP stable knockdown; 3) TGF-β signaling through β2SP/Smad3 significantly inhibits IGF2 transcription; ChIP assays validated binding of Smad3 at the human IGF2 promoter region between -859bp to -688bp upstream of the IGF2 transcriptional start site; 4) TCGA based integrated analyses identify specific HCC patient groups with altered TGF-β members and high expression of IGF2 that could be potentially targetable for IGF2; 5) TCGA analyses of 33 cancers also revealed that potentially “switching off” TGF-β-pathway (through decreased expression levels) leads to increased stemness validating findings of our TGF-β deficient mouse models that phenocopy a human cancer stem cell syndrome. In summary, TGF-β negatively regulates IGF2 transcription: loss of TGF-β signaling through β2SP/Smad3 leads to IGF2 activation, contributing to tumor progression. TGF-β-β2SP/Smad3 are important for IGF2 repression and maintaining stem cell homeostasis, which may result from TGF-β/β2SP/Smad3 and/or CTCF-dependent regulation of IGF2. Our studies provide a framework for new animal models of liver and gastrointestinal cancers and future new therapeutics.

Citation Format: Shuyun Rao, Sobia Zaidi, Kazufumi Ohshiro, Jian Chen, Shoujun Gu, Wilma Jogunoori, Jon White, Bibhuti Mishra, Shulin Li, Rehan Akbani, Lopa Mishra. Regulation of IGF2 by TGF-β signaling in liver cancers and stem cell homeostasis [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 5459.
Abstract 5464: Pharmacologic activation of REV-ERBs is lethal in cancer and oncogene-induced senescence
Authors: Sulli Gabriele , Rommel Amy , Wang Xiaojie , Kolar Matthew J. , Puca Francesca , Saghatelian Alan , Plikus Maksim V. , Verma Inder M. , Panda Satchidananda
Published: August 2018

The circadian clock drives daily rhythms in cell proliferation, metabolism, inflammation and DNA damage response. Perturbations of these processes are hallmarks of cancer, and circadian rhythm disruption predisposes to tumor development in animal models and humans. This raises the hypothesis that pharmacologic modulation of the circadian machinery may be an effective therapeutic strategy for combating cancer. The nuclear hormone receptors REV-ERBα and REV-ERBβ (REV-ERBs) are essential components of the circadian clock. Here we show that SR9009 and SR9011, two different agonists of REV-ERBs, are specifically lethal to cancer cells and oncogene-induced senescent (OIS) cells, including melanocytic naevi, while having no effect on viability of normal cells or tissues. Anticancer activity of SR9009 and SR9011 affects several oncogenic drivers (such as H-RAS, BRAF, PIK3CA, and others), and persists in the absence of p53 and under hypoxic conditions. The regulation of autophagy and de novo lipogenesis by SR9009 and SR9011 plays a critical role in evoking an apoptotic response in malignant cells. Importantly, the selective anticancer properties of these REV-ERB agonists impair glioblastoma growth in vivo and improve survival without causing any overt toxicity in mice. These results indicate that pharmacologic modulation of circadian regulators is an effective novel antitumor strategy, identifying the existence of a previously unknown class of anticancer agents with a wide therapeutic window. We propose that REV-ERB agonists are novel autophagy and de novo lipogenesis inhibitors with selective activity towards malignant and benign neoplasms.

Citation Format: Gabriele Sulli, Amy Rommel, Xiaojie Wang, Matthew J. Kolar, Francesca Puca, Alan Saghatelian, Maksim V. Plikus, Inder M. Verma, Satchidananda Panda. Pharmacologic activation of REV-ERBs is lethal in cancer and oncogene-induced senescence [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 5464.
Abstract 5480: Colorectal cancer cell lines harboring mutation in Wnt/β-catenin pathway shows enhanced sensitivity to the stearoyl-CoA desaturase (SCD) inhibitor, DSP-0692
Authors: Furuta Yudai , Fukunaga Yuichi , Ikeda Satoshi , Koga Erina , Umehara Hiroki , Otsubo Tsuguteru , Watanabe Manabu , Hasegawa Futoshi , Ban Hitoshi , Sugaru Eiji
Published: August 2018

Colorectal cancer (CRC) is one of the most frequent malignant diseases worldwide. Recent studies suggest that a small subpopulation of cells known as cancer stem cells (CSCs) may promote the high metastasis and relapse associated with CRC. It has been reported that Wnt/β-catenin signaling is aberrantly activated in a variety of cancer including colorectal cancer, and plays a critical role in CSC maintenance.

Here it is shown that a novel SCD inhibitor, DSP-0692, may inhibited Wnt/β-catenin signaling pathway through two different mechanisms in CRC. The effect of SCD inhibition on Wnt3A secretion, in which process post-translational fatty acylation (with palmitoleic acid) is required, was investigated. DSP-0692 led to reduced Wnt3A protein levels secreted in the media in Wnt3A-overexpressing cell line. This result was further confirmed by the TCF/LEF reporter assay in HCT116 incubated with conditional media from L-Wnt3A cells, with or without DSP-0692. DSP-0692 also inhibited exogenous Wnt3A-induced reporter activity. This indicates DSP-0692 may inhibit the β-catenin/Tcf transactivation by additional mechanism in addition to inhibiting Wnt secretion. To clarify this additional mechanism, the effect of DSP-0692 on nuclear translocation of β-catenin was investigated. SCD inhibition led to a decrease of β-catenin amounts within the nucleus. Currently, how SCD inhibition affects nuclear translocation of β-catenin is being investigated. It was confirmed that DSP-0692 exhibited lower IC50 value to sphere cells carrying mutations related to Wnt/β-catenin signaling pathway than those without these mutations. Next, the effect of SCD inhibition on Wnt/β-catenin signaling pathway in vivo was investigated. Treatment with DSP-0692 in a SW620 colorectal cancer xenograft model suppressed mRNA expression of the Wnt/β-catenin target genes, as well as CSC marker gene.

Taken together, these results suggest fatty acid desaturation in tumor cells may have an impact on Wnt/β-catenin signaling pathway and involve in CSC maintenance. The approaches targeting SCD could be a new therapeutic strategy for anti-CSC therapy in CRC.

Citation Format: Yudai Furuta, Yuichi Fukunaga, Satoshi Ikeda, Erina Koga, Hiroki Umehara, Tsuguteru Otsubo, Manabu Watanabe, Futoshi Hasegawa, Hitoshi Ban, Eiji Sugaru. Colorectal cancer cell lines harboring mutation in Wnt/β-catenin pathway shows enhanced sensitivity to the stearoyl-CoA desaturase (SCD) inhibitor, DSP-0692 [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 5480.
Abstract 5495: Inhibiting ERRα blocks liver steatosis and tumorigenesis induced by PTEN loss
Authors: Chen Chien-Yu , Li Yang , Chen Jingyu , He Lina , Stiles Bangyan
Published: August 2018

Mitochondrial dysfunction has been attributed to be a major cause for fatty liver development and also underlies tumor development. Using a liver-conditional Pten deletion model where the activation of its downstream PI3K/AKT signaling led to fatty liver, steatohepatitis, fibrosis and finally liver cancer development, we reported previously that the fatty liver and liver cancer is accompanied by elevated mitochondrial bioenergetics and a dramatic induction of estrogen-related receptor α (ERRα), a master regulator that orchestrate mitochondria response to modulate metabolism. In this project, we intended to investigate whether blocking ERRα expression and inhibiting mitochondrial respiration can attenuate tumor growth and lipid accumulation in the Pten null livers and human liver cancer cells. Using a genetic knockdown approach with siERRα and chemical genomic approach with a small molecular polyamide compound that binds to the ERRα consensus binding site, we showed that ERRα knockdown suppressed mitochondrial function and impeded cell proliferation in human liver as well as prostate cancer cell lines lacking Pten. In addition, blocking ERRα transcriptional activity with ERRα specific polyamide err-PA significantly reduced tumor growth in xenograft models. Furthermore, err-PA administration in vivo in 1.5-month old liver- specific Pten null mice remarkably prevented the development of fatty liver morphology and quantitatively reduced the hepatic triglyceride level. In summary, our study highlights ERRα's crucial role in regulating mitochondrial bioenergetics and underscores its therapeutic potential in cancer and lipid disorders.

Citation Format: Chien-Yu Chen, Yang Li, Jingyu Chen, Lina He, Bangyan Stiles. Inhibiting ERRα blocks liver steatosis and tumorigenesis induced by PTEN loss [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 5495.
Abstract 5502: Characterization of a hotspot PTEN mutation in endometrial cancer
Authors: Wang Ye , Jakubik Charles , Peterson Rachel , Benes Cyril , Haas Wilhelm , Engelman Jeffrey , Costa Carlotta
Published: August 2018

Endometrial cancer is the most common gynecologic malignancy in the developed world. These cancers harbor the highest rates of PI3K pathway alterations reported, with PTEN being the most frequently altered gene. Differently from other cancer types, in endometrial cancer a specific hotspot mutation in the lipid phosphatase domain of PTEN is present at residue 130. This hotspot mutation significantly co-occurs with other mutations in the PI3K pathway, such as PIK3CA and PIK3R1. Interestingly, in the TCGA data we also found a positive correlation between this mutation and the expression level of the estrogen receptor alpha. Although it has been reported that the PTEN-R130 mutant loses its lipid phosphatase activity in vitro, little is known regarding the other unconventional functions of PTEN, such as its protein phosphatase activity. We performed unbiased phospho-proteomics experiments in order to explore the impact of this specific mutation in multiple endometrial cancer cell models.

Citation Format: Ye Wang, Charles Jakubik, Rachel Peterson, Cyril Benes, Wilhelm Haas, Jeffrey Engelman, Carlotta Costa. Characterization of a hotspot PTEN mutation in endometrial 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 5502.
Abstract 5512: Dual inhibition of AXL and FN14 reverses cisplatin resistance in non-small cell lung carcinoma by inducing higher caspase 3 cleavage
Authors: Mukherjee Soumavo , Suresh Dhananjay , Zambre Ajit P. , Upendran Anandhi , Kannan Raghuraman
Published: August 2018

Background and Objectives: NSCLC patients undergoing Cisplatin therapy often develop resistance within 9-12 months that decreases treatment efficacy and is the major reason for the low median survival of 10-14 months among stage IV patients. Therefore, resistance is the main therapeutic limitation in the later part of the progressive disease. It has been suggested that suppressed Caspase-3 is one of the major reasons for failure to induce apoptosis by the cell during cisplatin resistance. We hypothesized this resistance to be related to EMT and other survival pathways. Recently, AXL and TWEAK/FN14 pathways have been shown to be upregulated in cisplatin resistant cells causing activation of EMT and survival pathways. Therefore, we examined whether dual inhibition of AXL and TWEAK/FN14 successfully reverses the cisplatin resistance in NSCLC.

Materials and Methods: Drug resistant EGFR mutant H1975 and KRAS mutant A549 cells were tested for cisplatin desensitization following knock down of AXL and FN14 inhibition. We studied resensitization by determining the IC50 values in the drug resistant cell lines by treatment with both AXL and FN14 siRNA. We investigated detailed cellular mechanisms imparting resistance by detecting relative levels of Caspase 3, cleaved Caspase 3, PARP, cleaved PARP, p-SRC, p-EGFR, AXL and FN14 by western blotting. Cytochrome-C induced apoptosis was detected by monitoring cytochrome-c released from mitochondria using fluorescence microscopy.

Results: Suppression of AXL and FN14 by siRNA treatment resensitized NSCLC to cisplatin and induced apoptosis. IC50 values were decreased significantly following dual inhibition (by more than 200%). A marked increase in cleaved caspase 3 levels was also observed in both mutant cell lines. A subsequent increase in PARP and cleaved PARP levels verified the fact that caspase 3 is indeed responsible for the apoptosis in these cells. Upregulation of p-SRC and eventually p-EGFR failed to suppress the apoptosis through PI3K/AKT pathway. Cytochrome c release from mitochondria further confirmed cellular apoptosis.

Conclusion: Our results show that dual inhibition of AXL and TWEAK/FN14 can reverse cisplatin resistance by inducing higher caspase 3 cleavage that can enhance survival of NSCLC patients undergoing chemotherapy.

Citation Format: Soumavo Mukherjee, Dhananjay Suresh, Ajit P. Zambre, Anandhi Upendran, Raghuraman Kannan. Dual inhibition of AXL and FN14 reverses cisplatin resistance in non-small cell lung carcinoma by inducing higher caspase 3 cleavage [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 5512.
Abstract 5525: Rgs8 and Rgs16 protect against pancreatitis and PDA progression
Authors: Choudhuri Shreoshi Pal , Zolghadri Yalda , Mascarenhas Luke , Wilkie Thomas
Published: August 2018

Pancreatic ductal adenocarcinoma (PDA) has the highest death rate among major cancers, and new treatments are desperately needed. The mice we developed in this study, KCR8-16, are an excellent mouse model for identification, characterization, and in vivo validation of novel PDA therapeutics. Kras oncogenic mutations (e.g., KrasG12D) are found in over 90% of human PDA. Kras can be activated by protein kinase and G-protein coupled receptor (GPCR) signaling. Regulator of G-protein signaling (Rgs) proteins regulate GPCR signaling by accelerating the GTPase activity of Gq- and Gi class alpha subunits. Activating alleles of Gq that are resistant to Rgs inhibition are found in benign precursors of PDA in humans. We previously reported that Rgs8 and Rgs16 are in vivo reporters of Kras activity in pancreatic intraepithelial neoplasia (PanIN), intraductal papillary mucinous neoplasm (IPMN), and PDA progression in KIC;Rgs16::GFP mice (p48::Cre/+; KrasG12D/+; Cdkn2af/f; Rgs16GFP ) (DMM 8, 2015). To identify the role of Rgs 8 and 16 in PDA, we crossed the Rgs8-16 double knockout into pancreas-specific KrasG12D (KC) mutant mice (termed KCR8-16). We found that deletion of Rgs8 and Rgs16 in KC accelerated PDA progression. Additional pancreatic stress evoked by caerulein treatment caused immediate and pancreas-wide progression to PDA in KCR8-16 mice. Our study suggests that Rgs8 and Rgs16 act as tumor-suppressor genes in PDA initiation and progression. Moreover, KCR8-16 and KIC;Rgs16::GFP mice can be used as an excellent model for identification and rapid in vivo validation of PDA therapeutics.

Citation Format: Shreoshi Pal Choudhuri, Yalda Zolghadri, Luke Mascarenhas, Thomas Wilkie. Rgs8 and Rgs16 protect against pancreatitis and PDA progression [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 5525.
Abstract 5545: Heterodimeric interleukin 15 (hetIL-15) treatment decreases primary breast cancer 4T1 tumors and alleviates the metastatic burden
Authors: Stellas Dimitris , Dimas Konstantinos , Nagy Bethany A. , Valentin Antonio , Felber Barbara K. , Pavlakis George N.
Published: August 2018

Introduction: In this preclinical study, we tried to model the therapeutic strategies often used in the clinic to treat breast cancer patients. We analyzed the role of hetIL 15 immunotherapy alone and in combination with chemotherapy and surgery using the 4T1, a triple-negative breast cancer cell line, murine breast cancer model.

Experimental Procedures: We evaluated the therapeutic potential of combining surgery, chemotherapy (doxorubicin) and immunotherapy (hetIL-15) in the murine 4T1 breast cancer model. Cancer cells were orthotopically implanted into the mammary fat pad of Balb/c mice, and the efficacy of hetIL-15 in combination with doxorubicin on the growth of primary tumors and metastatic disease was assessed. In addition, we monitored the impact of surgery alone in the development of metastatic disease, and, finally, we evaluated the effects on the metastatic burden of the three different approaches combined.

Results: hetIL-15 monotherapy and combination of doxorubicin chemotherapy with hetIL-15 immunotherapy inhibited the growth of primary tumors. Interrogating the role of these treatments on lung metastatic disease, the results were unexpected. Doxorubicin alone did not affect the numbers of the metastatic lesions in the lungs. In contrast, hetIL-15 monotherapy reduced the metastatic burden. The combination strategy did not show advantage as compared to hetIL-15 monotherapy. Surgical removal of the growing tumor also decreased metastatic burden, especially when performed at an early point. Chemotherapy with doxorubicin prior to and post-surgery had no impact on the metastatic disease. In contrast, hetIL-15 treatment strongly diminished or eliminated the metastatic burden.

Conclusion: Our data strongly suggest that treatment with hetIL-15 as an adjuvant therapy after surgery, or as a neoadjuvant agent before surgery, significantly impacts metastatic disease.

Citation Format: Dimitris Stellas, Konstantinos Dimas, Bethany A. Nagy, Antonio Valentin, Barbara K. Felber, George N. Pavlakis. Heterodimeric interleukin 15 (hetIL-15) treatment decreases primary breast cancer 4T1 tumors and alleviates the metastatic burden [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 5545.
Abstract 5547: Durable remission with nivolumab, anti-PD-1 monoclonal antibody, in a patient with advanced thymic carcinoma
Authors: Chae Young Kwang , Cho Anderson , Rhee Kyunghoon , Davis Andrew , Iams Wade , Bhave Manali , Cruz Marcelo , Park Lee Chun
Published: August 2018

Thymic carcinoma is a rare neoplasm with poor prognosis that forms on the outside surface of the thymus. Due to the rarity of the disease, there are no randomized clinical trials that guide therapy. Currently, there are clinical trials under way to assess the efficacy of anti-programmed death 1 (PD-1) monoclonal antibodies for the treatment of advanced thymic carcinomas. However, the role of PD-1 inhibitors in treating thymic carcinoma remains mostly unknown. Here we present a case of a patient with thymic carcinoma who showed durable remission when treated with nivolumab. 61-year-old male was diagnosed with thymic carcinoma during an episode of COPD exacerbation in April 2015. The patient was treated with neoadjuvant chemotherapy regimen consisting of four cycles of carboplatin and paclitaxel every three weeks from June to September 2015. The mass was resected in October 2015. Margins were found to be grossly positive. The patient was deemed a poor candidate for adjuvant chemotherapy, and in February 2016 the decision was made to begin 3 mg/kg nivolumab therapy considering the poor prognosis of disease and high risk of recurrence. Close to two years after initiating therapy, he currently has no measurable disease and tolerates the medication without side effects. This is the first report demonstrating durable remission of thymic carcinoma with nivolumab treatment. Our case suggests that prospective trials evaluating the use of nivolumab in patients with thymic carcinoma are warranted.

Citation Format: Young Kwang Chae, Anderson Cho, Kyunghoon Rhee, Andrew Davis, Wade Iams, Manali Bhave, Marcelo Cruz, Lee Chun Park. Durable remission with nivolumab, anti-PD-1 monoclonal antibody, in a patient with advanced thymic 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 5547.
Abstract 5548: The therapeutic potential of AMV564, a novel bispecific bivalent (2x2) T-cell engager, for the treatment of CD33-expressing hematologic malignancies
Authors: Han Tae H. , Guenot Jeanmarie , Denney William S. , Feldman Eric J.
Published: August 2018

AMV564 is a novel bispecific bivalent (2x2) immunotherapy that initiates an immunological synapse between T cells and CD33-expressing cells by binding both CD33 and CD3 with strong avidity. CD33 (Siglec-3) is a clinically-validated antigen target that is broadly expressed on the surface of malignant cells in acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and other hematologic malignancies, as well as on cells that potently suppress immune function, such as myeloid-derived suppressor cells (MDSC). AMV564 has been shown to eliminate MDSCs and restore hematopoiesis, induce potent cytolysis of leukemic cells in patient blood and bone marrow samples, and produce robust antitumor activity in a disseminated model of AML. In vitro studies, including cytotoxicity assays with AML patient samples, human T-cell activation assays, and cytokine-release assays, as well as animal safety studies were performed to characterize the pharmacokinetics, pharmacodynamics, and potential toxicities of AMV564. AMV564 demonstrated potent in vitro antitumor activity with EC50 values of approximately 0.7 - 3 pM. In vitro studies also demonstrated that leukemic cells are more sensitive to T-cell engagement using AMV564 than peripheral blood mononuclear cells by approximately 10-fold. In animal safety studies, AMV564 was tolerated at exposures that exceed those expected to have clinical antitumor activity by > 10-fold. Toxicological findings noted in these studies were anticipated based on the pharmacological activity of AMV564; observed findings included events related to CD3-mediated T-cell activation, cytokine release, and decreases in neutrophil and monocyte counts with rapid recovery. Results from a pharmacokinetic/pharmacodynamic model integrating the preclinical data suggest the potential for a wide therapeutic index for patients with AML. Based on the in vitro and in vivo preclinical data, AMV564 is expected to have a wide therapeutic index, with robust antitumor activity in AML and MDS at doses well below those predicted to be associated with dose-limiting toxicities, and a predictable and monitorable adverse event profile.

Citation Format: Tae H. Han, Jeanmarie Guenot, William S. Denney, Eric J. Feldman. The therapeutic potential of AMV564, a novel bispecific bivalent (2x2) T-cell engager, for the treatment of CD33-expressing hematologic malignancies [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 5548.
Abstract 5572: Circulating tumor cells: The tumor trail left in the blood
Authors: Kulasinghe Arutha , Perry Chris , Kenny Liz , Blick Tony , Warkiani Majid , Vela Ian , O'Byrne Ken , Thiery Jean-Paul , Thompson Erik , Nelson Colleen , Punyadeera Chamindie
Published: August 2018

Background: Metastasis in HNC patients is reflected by measurable levels of circulating tumor cells (CTCs) in the peripheral blood of cancer patients. CTCs represent cancer cells from the primary and metastatic sites, thereby providing a comprehensive representation of the tumor burden of an individual patient. For patients without CTCs at presentation, the detection of CTCs in the blood and analysis of biomarkers within them provide an opportunity to identify patients "at risk" of developing overt metastasis, accelerating targeted treatment in addition to routing care with the clear aim of improving cure.

Methods: Our study aimed to assess whether CTCs from the blood of HNC patients attending the Princess Alexandra Hospital and Royal Brisbane and Women's Hospital provided early cues of distant metastases (n=250).

Results: With significant advances in CTC isolation technologies, we could demonstrate a higher CTC capture efficiency using epitope-independent platforms. By assessment of single and clustered CTCs, our data showed that HNC patients can be identified 4-6 months prior to developing clinical/radiographically evident metastasis. In these patients, a window for treatment escalation could become a possibility. In a proof-of-principle study, using novel culture formulations and hypoxic conditions (1-2% O2), we were able to demonstrate, for the first time, short-term patient-derived CTC cultures ex vivo from 7/18 HNC samples (4/7 HPV-positive, oropharyngeal) in a clinically relevant time period. Recent advancements have shown that PD-1 immune checkpoint therapies have durable responses in metastatic HNC patients that fail 1st- and 2nd-line therapy. Our preliminary data suggest PD-L1 is frequently expressed on HNC CTCs, and an immunoscore may be able to stratify patients likely to respond to immunotherapy.

Conclusion: Expanding CTCs outside the patient's body allows for the recapitulation of the molecular diversity present within the tumor, understanding the disease progression and testing of therapies. Patients with a high percentage of PD-L1+ CTCs could be potential candidates for anti-PD-L1 therapy, a promising new immunotherapy.

Citation Format: Arutha Kulasinghe, Chris Perry, Liz Kenny, Tony Blick, Majid Warkiani, Ian Vela, Ken O'Byrne, Jean-Paul Thiery, Erik Thompson, Colleen Nelson, Chamindie Punyadeera. Circulating tumor cells: The tumor trail left in the blood [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 5572.
Abstract 5579: Increased number of EMT-like CTCs relative to epithelial CTCs reveals signatures of poor outcomes in pancreatic cancer
Authors: Zeinali Mina , Murlidhar Vasudha , Fouladdel Shamileh , Hafner Mathias , Shao Shimeng , Zhao Lili , Cameron Heather , Bankhead Armand Iii , Shi Jiaqi , Cuneo Kyle C. , Sahai Vaibhav , Azizi Ebrahim , Wicha Max S. , Simeone Diane M. , Nagrath Sunitha
Published: August 2018

Objective: Although circulating tumor cells' (CTCs) isolation from pancreatic adenocarcinoma (PaCa) patients is feasible, investigation of their clinical utility has proven less successful than other cancer types, due to poor technology sensitivity and EpCAM-only based CTC assays.

Design: We present an integrated technology and biology-based approach using a microfluidic “Carpet chip” to study the biologic relevance of rare CTCs. We isolate both circulating epithelial CTCs (EpCs) and EMT-like CTCs (EMTCs) simultaneously from whole blood of PaCa patients (n=35), targeting two different surface markers: anti-EpCAM and anti-CD133. Gene expression profiling of CTCs was performed with 96 genes (n=17).

Results: Recovery of cancer cell lines spiked into whole blood is ≥97% with >76% purity. Of the 35 PaCa patients analyzed, 34 had ≥5 EpCs/mL and 35 had ≥15 EMTCs/mL. Higher numbers of EMTCs were observed in patients with lymph node involvement (N1) compared to patients without. The ratio of EpCs to total number of CTCs marginally decreased with advanced stage, while the ratio of EMTCs increased. Gene expression profiling showed CXCR1 was significantly upregulated in EpCs whereas POU5F1/Oct-4 and MYC were upregulated in EMTCs, demonstrating inherent phenotypic differences. Early-stage patients with significantly better overall survival and/or progression-free survival showed significant higher expression of genes including ERCC1 and TTF1.

Conclusions: We demonstrated successful isolation of heterogeneous population of CTCs (EpCs and EMTCs). We recovered significantly higher number of EMTCs in PaCa patients compared to EpCs, reflecting aggressive nature of PaCa. Profiling of CTCs revealed genetic signatures relevant to predicting patients' outcomes.

Citation Format: Mina Zeinali, Vasudha Murlidhar, Shamileh Fouladdel, Mathias Hafner, Shimeng Shao, Lili Zhao, Heather Cameron, Armand Iii Bankhead, Jiaqi Shi, Kyle C. Cuneo, Vaibhav Sahai, Ebrahim Azizi, Max S. Wicha, Diane M. Simeone, Sunitha Nagrath. Increased number of EMT-like CTCs relative to epithelial CTCs reveals signatures of poor outcomes in pancreatic 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 5579.
Abstract 5585: Extracellular vesicle poly(A)+ RNA analysis for cancer biomarker screening
Authors: Murakami Taku , Yamamoto Cindy , Ogura Mieko , Oakes Melanie , Harada Hiroshi
Published: August 2018

Extracellular vesicles (EV) are a promising biomarker source and present in several biological fluids such as plasma (8.0 x 1011 particles/mL, 121 ± 2.3 nm in diameter) and urine (1.1 x 1011 particles/mL, 152 ± 11.2 nm in diameter). In order to streamline marker screening and validation in translational studies, we developed and optimized high throughput filter-based EV capture, poly(A)+ RNA isolation and gene expression assay protocols for RT-qPCR and RNA-seq. First, we confirmed that plasma and urinary EV mRNA profiles (12 to 15 genes by RT-qPCR) are stable up to 1 week at 4°C, and up to 5 repeats of freeze-thaw cycles, and urinary EV was stable up to 10 months at -80°C, indicating EV mRNA could be a useful biomarker source. Although EV mRNA is less abundant (550 pg mRNA from 1 mL plasma) compared to tissue mRNA or EV non-coding RNA, the optimized protocol allows quantification of about 9,000 genes in average by RNA-seq from 2 mL frozen plasma (N=3) with high reproducibility among the replicates (Spearman correlation: 0.9086 to 0.9326) and between the commercial EV kits (Spearman correlation: 0.9207 to 0.9683). About 77% of the 1000 most abundant genes in plasma EV are expressed ubiquitously throughout various organs but some are expressed specifically in blood (12%), spleen (4.4%), esophagus mucosa (1.2%), etc. according to the Genotype-Tissue Expression (GTEx) database. In urinary EV, other than ubiquitously expressed genes (88%), the genes specific to liver (1.5%), bladder (1%), brain (1%), etc. are present. These data suggest EV mRNA are released from various organs and circulating in biological fluids. The protocol was further applied to biomarker discovery for bladder cancer, and differential gene expression analysis of EV mRNA was conducted using bladder cancer (N=4), cancer remission (N=2), and healthy/disease control urine samples (N=4). Ingenuity pathway analysis indicated that cancer-related molecular and cellular functions are activated in bladder cancer urinary EV but not in remission, while immune-related functions are activated both in cancer and remission. These data indicate that EV mRNA is a promising biomarker source for cancer biomarker screening, and could be used in translational studies.

Citation Format: Taku Murakami, Cindy Yamamoto, Mieko Ogura, Melanie Oakes, Hiroshi Harada. Extracellular vesicle poly(A)+ RNA analysis for cancer biomarker screening [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 5585.
Abstract 561: Circulating DNA clean up and enrichment from liquid biological samples for rare biomarker detection
Authors: Lewis Samantha R. , Cowles Charles , Oostdik Kathryn , Knox Curtis , Hook Brad A.
Published: August 2018

A major roadblock to using circulating cell free DNA (ccfDNA) for cancer biomarker detection is the difficulty of obtaining a pure sample. Contaminating genomic DNA (gDNA) can overwhelm already rare events within a CCF sample and prevent sensitive detection of these key mutations. Unfortunately, liquid biological samples often are not collected and processed in an ideal manner, resulting in decreased quality and purity. Many studies report difficulties in detecting mutations in ccfDNA which may originate from variable levels of gDNA background based how samples are processed. To this end, we present an effective method for purifying relevant small nucleic acids from larger genomic species after DNA purification from plasma.

Using a simple magnetic bead based technique, we demonstrate efficient removal of gDNA even in highly contaminated samples. This method can be used to enrich liquid biological samples for improved detection of rare genetic events such as microsatellite instability and mutations in key oncogenes. Using plasma from three individuals with colorectal cancer tumors harboring known mismatch repair gene mutations, we compare detection of somatic mutations in ccfDNA before and after this gDNA clean up method. Microsatellite instability profiles were obtained from ccfDNA and compared to normal and tumor profiles to determine if evidence of mismatch repair abnormalities could be detected. Targeted amplicon sequencing was then performed to detect tumor associated mutations. DNA isolated from matched adjacent normal and tumor tissues were used as controls in these experiments to confirm mutations were of tumor not germline mutations.

Effectively removing gDNA contamination from ccfDNA samples can improve biomarker detection consistency from liquid biological samples. We demonstrate a magnetic bead-based method for ccfDNA clean-up, which can be used prior to microsatellite instability detection by fragment analysis or rare variant detection by next generation sequencing.

Citation Format: Samantha R. Lewis, Charles Cowles, Kathryn Oostdik, Curtis Knox, Brad A. Hook. Circulating DNA clean up and enrichment from liquid biological samples for rare biomarker detection [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 561.