bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1820: Circular RNAs contribute to neuroblastoma pathogenesis
Authors: Klironomos Filippos , Danssmann Clara , Naderi Julian , Winkler Annika , Luz Uschi , Hundsdoerfer Patrick , Eggert Angelika , Toedling Joern , Hertwig Falk , Schulte Johannes H. , Fuchs Steffen
Published: August 2019

Circular RNAs (circRNAs), a noncoding RNA class originating from alternative splicing, are highly abundant in neural tissues and can regulate gene expression by binding to and inhibiting microRNAs and RNA-binding proteins. We explored whether circRNAs influence pathogenesis in neuroblastoma, the most common solid extracranial tumor of childhood. We performed whole-transcriptome sequencing of primary neuroblastoma samples to identify candidate circRNAs, which were validated in a neuroblastoma cell line panel. Overexpression and knockdown models were created to investigate their impact on cell viability, proliferation, apoptosis and differentiation. We identified 4,482 unique circRNA backsplicing junctions involving 2,080 genes in 69 neuroblastoma samples from all risk groups. Candidate circRNA expression did not correlate with host gene expression, indicating independent regulatory mechanisms. Principal component analysis was conducted using tumor circRNA expression. MYCN-amplified tumors clustered tightly together, demonstrating that variations in circRNA expression identify MYCN-amplified neuroblastomas as do gene expression patterns. Comparing our RNA sequencing data with other cancers and healthy fetal brain tissue revealed a circRNA subset specifically upregulated in neuroblastoma that included a circRNA derived from the ARID1A tumor suppressor gene. Cell fractionization and RNA FISH localized circARID1A to the cytoplasm, where it could interact with miRNAs and RNA-binding proteins for functional consequences. Specific circARID1A knockdown reduced cell numbers and viability, and induced neurite outgrowth and differentiation markers. Neither knockdown, nor overexpression of circARID1A influenced ARID1A mRNA and protein levels. Ongoing efforts applying bioinformatics methods to investigate the mechanism of action have identified several binding sites of microRNAs and RNA-binding proteins enriched in the circARID1A sequence. We demonstrate that circRNAs are expressed in neuroblastomas unrelated to host gene expression, and that their expression can be used to cluster MYCN-amplified tumors. As one in a set of upregulated circRNAs, circARID1A acts in a tumor-promoting manner in neuroblastoma cell lines independent of the host gene.

Citation Format: Filippos Klironomos, Clara Danssmann, Julian Naderi, Annika Winkler, Uschi Luz, Patrick Hundsdoerfer, Angelika Eggert, Joern Toedling, Falk Hertwig, Johannes H. Schulte, Steffen Fuchs. Circular RNAs contribute to neuroblastoma pathogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1820.
Abstract 1822: A mitochondrial pfeRNA associates with Far Upstream Element Binding Protein 1 (FUBP1) to promote lung adenocarcinoma tumorigenesis
Authors: Fackche Nadege T. , Mei Yuping , Ito Tomoaki , Garner Matthew , Brock Malcolm
Published: August 2019

Background: Protein Functional Effector RNAs (pfeRNAs) are novel small non-coding RNA molecules with great prognostic and diagnostic potential for NSCLC. They feature direct binding to, and regulation of phosphorylated proteins involved in lung cancer tumorigenesis pathways. Using next-generation sncRNA deep sequencing, we have identified a mitochondrial pfeRNA (mtpfeRNA) that could play a key role in tumorigenesis and cancer development of lung adenocarcinoma (ADC).

Methods: Using next-generation sncRNA deep sequencing, we analysed ADC and human normal bronchial epithelial (HBE) cell lines; and biospecimens including matched biopsy-proven ADC tissue, histologically normal adjacent lung tissue, benign lymph node, and (iv) malignant lymph nodes; Identifying a mitochondrial pfeRNA that could play critical roles in lung cancer tumorigenesis. Next, we examined the impact of this mtpfeRNA on lung ADC cell line viability. Liquid chromatography-tandem mass spectrometry analysis and reverse immunoprecipitation assays were used to identify critical interacting protein partners known to drive tumorigenesis. We further correlated our findings to clinical staging of ADC tumor cores by IHC staining.

Results: The expression levels of mtpfeRNA were upregulated in ADC compared to that of squamous cell carcinoma and normal human bronchial epithelial cells. We also noted that blocking the upregulated mtpfeRNA resulted in significant decreases in cell viability in ADC cells. Next, we determined that mtpfeRNA binds to Far Upstream Element Binding Protein 1 (FUBP1). Furthermore, we confirmed that both mtpfeRNA and FUBP1 expression correlated to stages and lymph node metastasis in patients with ADC.

Conclusion: We revealed novel functions of a mtpfeRNA in ADC, providing potential molecules for developing diagnostics and targeted therapy to improve the dismal survival rate of patients with ADC.

Citation Format: Nadege T. Fackche, Yuping Mei, Tomoaki Ito, Matthew Garner, Malcolm Brock. A mitochondrial pfeRNA associates with Far Upstream Element Binding Protein 1 (FUBP1) to promote lung adenocarcinoma tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1822.
Abstract 1826: MALAT1 regulates EGFR translation by KTN1-mediated transcriptomic change in cutaneous squamous cell carcinoma
Authors: Gao Lin , Zhou Liang , Li Shanshan , Ding Zhenhua , Qiu Junying , Hong Malin , Zou Chang
Published: August 2019

Introduction MALAT1 is a member of lncRNAs that has been implicated in breast, cervical, colorectal cancers. However, its role in cutaneous squamous cell carcinoma (cSCC) is not clear. In this study, functional studies using in vitro and in vivo confirmed MALAT1 promotes tumor growth, migration and invasiveness of cSCC. MALAT1 regulates EGFR protein expression but not in RNA level. It shows that identified KTN1 as the key mediator for MALAT1 regulation of EGFR.

Methods Total RNAs from cells were extracted using TRIzol for qRT-PCR. Cells were transfected with MALAT1 ASOs or siKTN1_1, siKTN1_2 and cell proliferation were detected using a cell counting kit as described in the manual. Cells were seeded into chambers for transwell assay. Visible cells were photographed and counted after stained for cell migration and invasiveness assays. Total cell or cSCC tissues were prepared and assayed by western blot as previously described. Bound antibodies were visualised using the Luminated Forte Western HRP substrate. For histochemistry staining, 10 μm tissue sections were fixed for 10min with 4% paraformaldehyde, and then incubated with antibodies. The pictures of stained IHC sections were taken under Leica Fluorescence Microscope. Statistical data were presented as mean values ± stand deviation and considered to reflect significant differences.

Results Our results revealed that MALAT1 was markedly higher expressed in A431, HSC-1 and HSC-5 cSCC cells than in HaCaT keratinocytes. MALAT1 exhibited much higher expression in cSCC primary tumors compared to normal tissues. In addition, KTN1 and EGFR protein were higher expression in cSCC tissues. Significant loss of MALAT1 repressed proliferation, migration, and invasiveness but promoted apoptosis of cSCC cells. Loss of MALAT1 led to the significant downregulation of KTN1 through RNA sequencing assay accompanied with lower EGFR protein expression. However, knockdown of KTN1 had effect on the protein expression of EGFR but no effect on RNA levels. The results implied that MALAT1 may regulate EGFR protein expression through inducing KTN1 mRNA transcription.

Conclusions In this study, we revealed a novel mechanism of cSCC treatment, MALAT1 can transcriptionally activates KTN1, positively regulates EGFR protein expression in cSCC. It may provide a new series of drug target for anti-cSCC therapy.

Citation Format: Lin Gao, Liang Zhou, Shanshan Li, Zhenhua Ding, Junying Qiu, Malin Hong, Chang Zou. MALAT1 regulates EGFR translation by KTN1-mediated transcriptomic change in cutaneous squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1826.
Abstract 1831: Role of autophagy in lymphangiosarcoma initiation and progression
Authors: Yang Fuchun , Sun Shaogang , Guan Jun-Lin
Published: August 2019

Lymphangiosarcoma is a rare malignant vascular tumor which is formed from aberrant proliferation of endothelial cells (ECs). We previously established a vascular tumor mouse model by conditional knockout of Tsc1 gene in ECs and revealed that hyper-activated mTORC1 and increased VEGF autocrine are required for lymphangiosarcoma development and progression. However, we also found that there are other unknown factors which contribute to vascular tumor development besides mTORC1 hyper-activation. Autophagy has been shown to play an important role in tumor initiation and progression. However, the role of autophagy in mTORC1-dependent vascular tumor development is unclear. In this study, we studied the potential role and mechanism of autophagy in promoting lymphangiosarcoma development. FIP200 (FAK family interacting protein of 200 kDa) was identified as an essential autophagy gene, which was shown to play a key role in breast cancer development and progression. By crossing Tsc1f/f, Scl-Cre mice with Fip200f/f mice, we generated Tsc1/Fip200 double cKO mice. We found that Fip200 deletion blocked lymphangiosarcoma development induced by mTORC1 hyper-activation in mice. We established an immortalized lymphangiosarcoma cell line named Tsc1ΔEC from Tsc1 cKO mice and examined their growth in xenograft transplant models following CRISPR-Cas9 knockout of Fip200, Atg5 or Atg7 in Tsc1ΔEC cells. Our data showed that KO of each of these autophagy genes impaired vascular tumor growth in nude mice. Mechanistically, we found that Fip200 KO suppressed mTORC1 signaling activation under glucose starvation condition. In addition, we found that both Fip200 KO and lipase inhibitor Orlistat treatment in Tsc1ΔEC inhibited mitochondria respiration and decreased ATP content by seahorse assay. We also found that deletion of Fip200, Atg5, Atg7 or LAL in Tsc1ΔEC blocked lipid droplets (LDs) degradation and decreased glycerol release. Collectively, our data suggest that autophagy is required for lymphangiosarcoma initiation and progression through mTORC1 hyper-activation maintenance under stress conditions and supplying energy consumption by regulating lipid metabolism.

Note: This abstract was not presented at the meeting.

Citation Format: Fuchun Yang, Shaogang Sun, Jun-Lin Guan. Role of autophagy in lymphangiosarcoma initiation and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1831.
Abstract 1832: Ligand-dependent Wnt signaling directs metabolic adaptations to promote pancreatic tumorigenesis
Authors: Aguilera Kristina Y. , Riahi Rana , Saadat Edris A. , Lay Anna R. , Le Thuc , Cabebe Anthony , Donahue Timothy R. , Radu Caius , Dawson David W.
Published: August 2019

Wnt/β-catenin signaling plays important roles in pancreatic adenocarcinoma (PDA) tumor initiation and progression via broad-ranging effects on proliferation, differentiation, survival, and stemness. Although Wnt has been shown to promote glycolysis in colon cancer, its role in regulating metabolism in PDA has not been explored in any detail. For this study, the AsPC-1 cell line was selected as an exemplar of ligand-dependent autocrine Wnt signaling in PDA. AsPC-1 harbors an inactivating mutation in RNF43 that confers Wnt growth-dependency and response to LGK974, a PORCN inhibitor that blocks Wnt ligand secretion and signaling. Global transcriptomic, proteomic, and metabolomic analysis revealed several metabolites and putative mediators of metabolism that are directly or indirectly linked to autocrine Wnt signaling in AsPC-1. In contrast to colon cancer, pharmacologic inhibition of Wnt by LGK974 in AsPC-1 led to the accumulation of pyruvate and lactate, as well as reduced tricarboxylic acid cycle metabolites and decreased mitochondrial membrane potential as measured by tetramethylrhodamine assay. These metabolic changes were accompanied by cell cycle arrest and downregulation of transcriptional programs involved in cell cycle progression, DNA replication, and nucleotide metabolism. Pyruvate analogs dichloroacetate and oxamate, that respectively target pyruvate dehydrogenase kinase and lactate dehydrogenase, restored mitochondrial membrane potential in the context of LGK974. Importantly, LGK974 results were phenocopied by siRNA-mediated knockdown of WNT7B. Thus, inhibition of mitochondrial oxidative phosphorylation by LGK974 in RNF43-mutant PDA appears mechanistically linked to its inhibition of canonical Wnt signaling, an adaptation and potential metabolic vulnerability that could be leveraged to therapeutic advantage via combinatorial strategies. Altogether, these results suggest tissue context and differing mechanisms of Wnt pathway activation or signal strength may profoundly influence its role in regulating cancer metabolism.

Citation Format: Kristina Y. Aguilera, Rana Riahi, Edris A. Saadat, Anna R. Lay, Thuc Le, Anthony Cabebe, Timothy R. Donahue, Caius Radu, David W. Dawson. Ligand-dependent Wnt signaling directs metabolic adaptations to promote pancreatic tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1832.
Abstract 1844: xCT promotes tumor formation in airway epithelial cells by activation MYC and WNT pathways
Authors: Ji Xiangming
Published: August 2019

INTRODUCTION: Many tumors increase uptake and dependence on nutrients such as glucose, cysteine or glutamine. These basic observations on cancer cell metabolism have opened multiple new diagnostic and therapeutic avenues in cancer. Recent studies demonstrated that smoking can induce the expression of xCT (SLC7A11) in normal epithelial cells and cancer cells, suggesting that overexpression of xCT may support lung tumor progression. These basic observations on cancer cell metabolism have opened multiple new diagnostic and therapeutic avenues in cancer. Here, we investigated the function of SLC7A11 (xCT), a cystine/glutamate anti-porter, in the pathogenesis of lung cancer. Recently, we found xCT to be strongly overexpressed at the cell surface of lung cancers and began to investigate the mechanisms by which xCT may contribute to lung cancer progression.

METHODS: We overexpressed the xCT in mutiple non-tumorgenic cell lines. These cell lines included 16HBE, BEAS2B, HBEC_KTR_P53_C7, and HEK293. We further investigated the role of xCT in cell proliferation, conly formation, and tumor formation in nude mice. Finally, we used RNAseq analysis to indentify the molecular mechanims associated with xCT expression in HEK293 cells.

RESULTS: Our results shows that xCT overexpression transformed the normal airway epithelium cell lines (HBEC3_KTR_P53_C7). The overexpression of xCT in HBEC_KTR_P53C7 induced colony formation in soft agar and promoted cell proliferation. In addition, we found overexpression of xCT reprogrammed multiple metabolic pathways such as glycolysis, glutaminolysis, and pentose phosphate pathway. In addition, we found overexpression of xCT could transform non-tumorigenic cell line HBEC_KTR_P53_C7 to tumor-formation cell line. Our RNAseq data indicated that this transformation is enriched in MYC and WNT pathway.

CONCLUSION: Our results demonstrate that xCT is highly expressed in a subset of NSCLCs. The induction of xCT in transformed aiway epithelial cells causes tumor formation in nude mice. The mechanism of malignant transformation involves the Warburg effect. Further investigation into the mechanisms of transformation may help in answering fundamental questions pertaining to how glutamate/cystine exchange contributes to lung cancer development and progression and also in establishing xCT as a potential novel therapeutic target in lung cancer.

Key words: Non-small cell lung cancer, amino acid transporters, metabolic reprogramming

Note: This abstract was not presented at the meeting.

Citation Format: Xiangming Ji. xCT promotes tumor formation in airway epithelial cells by activation MYC and WNT pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1844.
Abstract 1848: The role of PDK1 in metabolic reprogramming of mesenchymal glioblastoma
Authors: Jain Neha , Guda Maheedhara , Labak Collin , Smith Chase , Park Simon , Tsung Andrew , Velpula Kiran
Published: August 2019

Despite multimodal therapy and ever-improving surgical technique, glioblastoma (GBM) maintains its poor prognosis due to the chemotherapeutic resistance it builds over time, eventually rendering existing agents ineffective. A well-characterized phenomenon that takes place particularly in mesenchymal GBM tissue is the Warburg Effect, characterized by a shift from oxidative phosphorylation to glycolysis as a primary means to derive energy. Pyruvate Dehydrogenase Kinase 1 (PDK1) is one upregulated molecule in this shift toward anaerobic metabolism that is known for phosphorylating pyruvate dehydrogenase, the “gate keeper” of the TCA cycle. We sought to characterize further the role of PDK1 in terms of its metabolic role and any extra-metabolic functions it may serve. By silencing PDK1 and considering changes in downstream expression, we hope to understand what other factors serve as contributors to metabolic alterations seen in GBM. Elucidating the mechanism of this crucial shift more thoroughly, especially without the prominently cited role of PDK1, can begin to expose new targets for interrupting tumor maintenance and proliferation by affecting its metabolism. In this study we used differential expression of two vectors in which PDK1 was silenced, shPDK1 and SCBT-PDK1. GSEA analysis was done and analyzed to identify classes of genes or proteins that are over-represented and to identify significantly enriched or depleted groups of targets whose expression was vastly changed by silencing of PDK1. Using GSEA heat map data, gene targets whose expression was noted to be consistently changed with silencing of PKD1 targets were identified. Datamining and immunoblot analyses were then conducted to evaluate for expression of these genes in correlating GBM specimens. Ultimately, we further characterize PDK1’s global role in GBM and evaluate non-metabolic effects of PDK1 that may still affect survival and proliferation of GBM tumors.

Citation Format: Neha Jain, Maheedhara Guda, Collin Labak, Chase Smith, Simon Park, Andrew Tsung, Kiran Velpula. The role of PDK1 in metabolic reprogramming of mesenchymal glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1848.
Abstract 186: Androgen deprivation promotes neuroendocrine differentiation and angiogenesis through CREB-EZH2-TSP1 pathway in prostate cancers
Authors: Zhang Yan , Zheng Dayong , Zhou Ting , Hulsurkar Mohit , Ittmann Michael , Shao Long , Gleave Martin , Li Wenliang
Published: August 2019

The incidence of aggressive neuroendocrine prostate cancers (NEPC) related to androgen-deprivation therapy (ADT) is rising. NEPC is still poorly understood, such as its neuroendocrine differentiation (NED) and angiogenic phenotypes. Here we reveal that NED and angiogenesis are molecularly connected through EZH2 (enhancer of zeste homolog 2). NED and angiogenesis are both regulated by ADT-activated CREB (cAMP response element-binding protein) that in turn enhances EZH2 activity. We also uncover anti-angiogenic factor TSP1 (thrombospondin-1, THBS1) as a direct target of EZH2 epigenetic repression. TSP1 is downregulated in advanced prostate cancer patient samples and negatively correlates with NE markers and EZH2. Furthermore, castration activates the CREB/EZH2 axis, concordantly affecting TSP1, angiogenesis and NE phenotypes in tumor xenografts. Notably, repressing CREB inhibits the CREB/EZH2 axis, tumor growth, NED and angiogenesis in vivo. Taken together, we elucidate a new critical pathway, consisting of CREB/EZH2/TSP1, underlying ADT-enhanced NED and angiogenesis during prostate cancer progression.

Citation Format: Yan Zhang, Dayong Zheng, Ting Zhou, Mohit Hulsurkar, Michael Ittmann, Long Shao, Martin Gleave, Wenliang Li. Androgen deprivation promotes neuroendocrine differentiation and angiogenesis through CREB-EZH2-TSP1 pathway in prostate cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 186.
Abstract 1885: CRISPR/Cas9 engineered immortalized breast epithelial MCF10A reporter line for EMT studies and anti-cancer drug discovery
Authors: Shu Weiguo , Kumari Sangeeta , Douglas Diana , Rodriguez Luis G. , Newman Robert
Published: August 2019

Metastasis is responsible for most cancer-related deaths. One model for the mechanism of metastasis involves epithelial-to-mesenchymal transition (EMT), a process characterized by the decrease in cell adhesion and increase in cell motility. Cells undergoing EMT often display downregulation of epithelial markers (such as, E-cadherin) and upregulation of mesenchymal markers (such as, Vimentin). Besides metastasis, EMT has also been reported to be associated with other pathological conditions, such as acquired therapeutic drug resistance. Given the roles that EMT may play in these various pathological processes, it is of increasing interest as a target for anti-cancer treatment and drug discovery. In vitro reporter models have proven to be a valuable tool for dissecting the signaling pathways that regulate the EMT process and for screening compounds targeting EMT. In previously developed EMT reporter cell lines, the reporter gene was driven by a truncated EMT marker gene promoter. Therefore, the establishment of a more physiologically relevant reporter cell models is critical for advancing our knowledge of EMT. E-cadherin, a hallmark of epithelial cells, has been implicated in the onset of metastatic dissemination. Utilizing CRISPR/Cas9 knock-in technology, we have generated an E-cadherin (ECAD)-emerald green fluorescent protein (EmGFP) reporter model using immortalized breast epithelial MCF10A cells. In the reporter cells, EmGFP gene was tagged at the C-terminus of E-cadherin, allowing for real-time monitoring of EMT progression in live cells. The targeted knock-in of ECAD-EmGFP allele was verified at the genomic DNA, transcript (mRNA), and protein levels. Bio-functional evaluation of the reporter cell line revealed that treatment of ECAD-EmGFP reporter cells with TGFβ led to EMT induction, as demonstrated by a reduction in E-cadherin-GFP expression and increase in vimentin and fibronectin expressions. Additional functional characterization revealed that the reporter cells possessed an enhanced migration capacity upon EMT induction with TGF-β. In summary, this MCF10A-Ecad-EmGFP reporter cell line can serve as a physiologically relevant in vitro cell model for studying EMT cancer biology and anti-EMT drug discovery.

Citation Format: Weiguo Shu, Sangeeta Kumari, Diana Douglas, Luis G. Rodriguez, Robert Newman. CRISPR/Cas9 engineered immortalized breast epithelial MCF10A reporter line for EMT studies and anti-cancer drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1885.
Abstract 1890: Calcitonin receptor is required for T-antigen-induced prostate carcinogenesis
Authors: Aldahish Afaf , Thakkar Arvind , Shah Girish V.
Published: August 2019

Calcitonin & calcitonin receptor axis (CT-CTR) expression is increased in prostate cancer & its levels positively correlate with Gleason grade of prostate cancer. Moreover, activation of CT-CTR autocrine axis promotes several processes with tumor progression such as, tumor growth, invasion, angiogenesis, chemoresistance & metastasis. However, the role of CT-CTR axis in carcinogenesis process has not been investigated. To examine the role of CT-CTR axis in prostate carcinogenesis we examined the impact of CTR deficiency on T-antigen-induced prostate tumorigeneses in transgenic mice. We achieved this by cross-breading CTR-knockout mice with LPB-Tag mice (the transgenic mice that uses long Probasin promotor for prostate-specific expression of Tag T-antigen). The cross-breeding of these two transgenic mice yielded four different groups of mice with the following genotypes 1. Control (which lacked both transgenes), 2. CTRKO (only CTRKO transgene), 3. LPB-Tag (contained LPB-Tag transgene), & 4. CTRKO-LPB-Tag (expressed Both CTRKO & LPB-Tag transgenes). The growth of these mice were monitored until they reached the age of 90 days. The mice were sacrificed, the prostates were collected, fixed & evaluated by histology & immunohistochemistry. The results suggest that the mice of all groups developed normally with the exception of LPB-Tag group which displayed slower growth & the presence of prostatic tumors at the time of necropsy. Although all LPB-Tag mice displayed all prostate tumors, as expected wild type & CTRKO mice did not display change in the size or mass of prostates. Subsequently, H&E histology as well as Ki67 staining confirmed that all LPB-Tag mice displayed well-developed prostatic adenocarcinoma, all CTRKO-LPB-Tag displayed no presence of tumor with the exception of occasional presence of HGPIN. Since our earlier studies have shown that CTR induced epithelial-to-mesenchymal transition (EMT) in prostate cancer cell lines, we also examined the expression of two epithelial markers, ZO-1 & E-cadherin, & two mesenchymal markers, fibronectin & vimentin. The results suggest that prostates of the three groups ( WT, CTRKO, LPB-Tag-CTRKO) displayed higher epithelial markers expression with a remarkably lower expression of mesenchymal markers when compared with those of LPB-Tag group. This was further substantiated by the expression of EMT markers such as snail & N-cadherin. In conclusion, this study shows that the CTR deficiency inhibits prostate carcinogenesis in LPB-Tag mice without reducing T-antigen expression or causing any other apparent harmful side effects. This work was supported by NIH grant CA096534.

Citation Format: Afaf Aldahish, Arvind Thakkar, Girish V. Shah. Calcitonin receptor is required for T-antigen-induced prostate carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1890.
Abstract 191: Extracellular signaling linked to PI3K/AKT triggers the formation of vasculogenic mimcry in ovarian cancer
Authors: Valdivia Andres , Mingo Gabriel , Aldana Varina , Sandoval Alejandra , Corvalan Alejandro , Nualart Francisco , Owen Gareth I.
Published: August 2019

Introduction: The presence of an endothelial cell-free perfusion pathway, known as Vasculogenic Mimicry (VM), strongly correlates with reduced patient survival, however the mechanisms by which a tumor can create a self-generated irrigation system remain unknown. Currently there is no mechanism of VM formation reported in fluid conducting tubular structures. Herein, using a standardized in vitro model, we sought to understand the pathways required for VM formation.

Methods: We utilized confocal microscopy and Imaris reconstruction to demonstrate that cancer cells can create an internal tubular network in cultured cancer cell lines, cancer spheres and primary cancer cultures of ovarian cancer. Matrigel and 3D matrices containing specific protein components allowed the deciphering of the extracellular matrix proteins involved in VM formation. Intercellular pathways were resolved using chemical inhibitors.

Results: VM only occurs when cells are grown in 3D culture and only 30% of primary cancer cultures undergo this process. The presence of laminin 1 is sufficient to trigger the formation of tubular structures and this may involve the alpha2beta1 integrin and metalloproteases. Downstream FAK and PI3K/AKT intercellular pathways are required to form functional tubular networks. Microarray data elucidated the presence of the mRNA transcripts that were increased in cells that undergo VM.

Discussion: We demonstrate that Laminin 1, a component of the extracellular matrix (ECM), can promote the process of VM, which further involves the downstream signaling of the potentially druggable targets of the PI3K pathway. As VM is associated with reduced patient survival, elucidation of the mechanisms of formation may deliver new cancer therapies.

Funding: FONDECYT 1180241, CONICYT FONDAP 15130011, IMII P09/016F

Citation Format: Andres Valdivia, Gabriel Mingo, Varina Aldana, Alejandra Sandoval, Alejandro Corvalan, Francisco Nualart, Gareth I. Owen. Extracellular signaling linked to PI3K/AKT triggers the formation of vasculogenic mimcry in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 191.
Abstract 1910: Regulation of ovarian tumor microenvironment dynamics by compressive stress
Authors: Asem Marwa , Young Allison Young , ClaureDeLaZerda Alejandro , Ravosa Matthew , Stack Sharon
Published: August 2019

Ovarian cancer (OvCa) is the most fatal gynecologic malignancy and the fifth leading cause of overall cancer death among American women with a low (27%) 5-year survival rate, as 75% of women are diagnosed with disseminated intra-peritoneal (IP) metastasis. OvCa cells detach from the primary tumor and shed into the peritoneal cavity, adhere to the peritoneal mesothelial cell (MC) monolayer, intercalate within this layer, and invade into the submesothelial matrix, where they proliferate and form secondary lesions.

More than one third of OvCa patients develop ascites that correlates with relapse and poor prognosis. The accumulated ascites can reach volumes up to 5 liters, causing a striking elevation in intraperitoneal pressure (IPP) from normally sub-atmospheric ~5 mmHg to as high as 27 mmHg, impacting the tumor microenvironment including host and tumor cells. The pressure created by accumulated ascites alters the force environment in the peritoneal cavity, causing strain and compressive forces on peritoneal structures. Yet, the mechanism through which ascites-induced IPP influences OvCa progression is poorly understood. The aim of this study is to understand how ascites-induced IPP promotes OvCa progression.

To model the compressive force present in vivo due to accumulated tense ascites (~27 mmHg), we evaluated the effects of compressive force on primary human and murine MC monolayers and murine peritoneal explants using the Flexcell Compression System. Strikingly, compressed MC and peritoneal explants exhibited phenotypic alterations and formed abundant Tunneling Nanotubes (TNT), which are cell membrane extensions that modulate transfer of organelles between cells under stress. Furthermore, compression of OvCa cells adherent to murine peritoneal explants show intense formation of TNT between OvCa cells and peritoneal MC. Remarkably, TNT formed under compression displayed membrane distension and modulated the transfer of mitochondria from MC to OvCa cells. Moreover, collected peritoneal explants from OvCa murine model with intense ascites showed TNT formation and changes in the peritoneal sub-mesothelial collagen ultrastructure.

This study reveals a novel response of OvCa cells and the tumor microenvironment to mechanical compression, uncovering a new mechanism by which OvCa metastatic success may be regulated.

Note: This abstract was not presented at the meeting.

Citation Format: Marwa Asem, Allison Young Young, Alejandro ClaureDeLaZerda, Matthew Ravosa, Sharon Stack. Regulation of ovarian tumor microenvironment dynamics by compressive stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1910.
Abstract 1914: Acute myeloid leukemia cell and stem-progenitor cell behavior studied in mimetic bone marrow microenvironment
Authors: Simonetti Giorgia , Malferrari Marco , Becconi Maila , Bruno Samantha , Martinelli Giovanni , Rapino Stefania
Published: August 2019

The bone marrow (BM) microenvironment plays a pivotal role in the survival and chemoresistance of leukemia stem cells (LSC) in acute myeloid leukemia (AML). However, conventional culture approaches do not reflect physiological conditions of crucial anatomical districts and signals of incubation-induced dysregulation may confound leukemic transcriptome results. In vivo mimetic cultures of the LSC districts will significantly aid in the disentanglement of LSC mechanisms and in the assessment of the therapy. We engineered Petri dishes able to mimic the “hypoxic” conditions found in the stem cell niches and in BM district. Our approach is based on a patented method which controls the physiological concentrations of oxygen, glucose and pH in the culture environment. Cell proliferation and hypoxia-induced apoptosis in the engineered device were investigated for OCI-AML3 and KG1 AML cell lines. We observed a slight but significant reduction in cell proliferation compared with control cells for OCI AML-3, which is in line with a more quiescent cell condition. No significant differences were obtained by culturing KG-1 cells, which are more resistant to low oxygen concentrations. We have analyzed the effects of cell culturing in the hypoxic dishes on the expression of MYC and p21. Immunofluorescence analysis of HIF-1α levels and HIF-1α nuclear localization as a function of oxygen concentration were determined. Time-lapse experiments of AML cell lines and primary BM cells were performed in the in vivo mimetic device to elucidate the response of CD34+ AML cells to different oxygen concentrations. We tested an innovative cell culturing device able to mimic the dynamical conditions of oxygen content, which are found in vivo. Our results shed light on AML cells and progenitor-stem cells behavior in response to oxygen conditions in the BM niche and allow to disentangle the LSCs complex biology. This ”in vivo mimetic” approach will generate crucial information on the molecular mechanisms preserving LSC in AML, thus providing novel potential therapeutic targets to eradicate leukemia initiating cells in their own microenvironment, contrasting therapy resistance.

Supported by: Associazione Italiana per la Ricerca sul Cancro, MFAG id. 19044.

Citation Format: Giorgia Simonetti, Marco Malferrari, Maila Becconi, Samantha Bruno, Giovanni Martinelli, Stefania Rapino. Acute myeloid leukemia cell and stem-progenitor cell behavior studied in mimetic bone marrow microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1914.
Abstract 1927: High-throughput cell line panel drug screening in organoids and 3D systems
Authors: Scales Tim M. , Rada-Kovacs Anett , Pemberton Helen N. , Wiggins Ceri M. , McCarthy Nicola J. , Little Annette S. , Sorrell David A.
Published: August 2019

Cell panel screening of cancer lines grown in standard 2D conditions is a well-established component of the drug discovery toolbox. A sufficiently large cell line panel, reflecting the molecular heterogeneity within a cancer type/sub-type, is required to detect correlations with robust statistical power that aid biomarker identification and patient stratification. However, the heterogenous exposure of tumor cells to nutrients, oxygen and other physical or chemical stresses found in three-dimensional solid tumors also has a role to play in patient response and this is not well mimicked in 2D monolayer cell cultures.

To meet the need for high-throughput cell-panel screening in more complex culture systems, we have developed 3D cell-based screens to more closely model the complex physiological environment found in tumors. We have characterised our OncoSignature panel of 300 cell lines for growth as spheroids to produce a 200 strong panel of spheroid forming lines. Using our high-throughput platform to compare drug activity in 2D and 3D cultures, we have identified clear differentials for some compounds in single agent and drug combination screens. For example, MEK and EGFR inhibitors have significantly higher activity in 3D cultures than 2D cultures, with a greater than 10-fold shift in EC50 values. These results were validated in 3D soft agar assays, with similar compound potencies being observed in the HTS spheroid assay and the lower throughput soft-agar assay.

We have further extended our 3D screening into organoid cultures. Organoid cultures, with increased complexity in terms of structure and cell heterogeneity compared to spheroids, have historically been challenging to use in high-throughput screening. However, our initial results show encouragingly robust data, with compound activity that is congruent with the genetic characteristics of the organoids.

Finally, we used our integrated screening platform to evaluate the power of CRISPR approaches to examine genetic dependencies in KRAS mutant cells in 2D, 3D spheroid and organoid cell cultures. Pooled, next-generation sequencing-linked CRISPRko allows an alternative and complementary genomics approach to arrayed cell panel screening, providing a powerful and robust target identification and validation strategy.

Overall, Horizon’s 3D screening platforms will enable large scale interrogation of both drug and gene interactions in more complex cell culture systems.

Citation Format: Tim M. Scales, Anett Rada-Kovacs, Helen N. Pemberton, Ceri M. Wiggins, Nicola J. McCarthy, Annette S. Little, David A. Sorrell. High-throughput cell line panel drug screening in organoids and 3D systems [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1927.
Abstract 1933: Tissue print Vmem imaging: Visualizing bioelectric signatures in cancer
Authors: Adams Dany S. , Tracey Brian H. , Takiff Larry , Kearns James , Naber Stephen P. , Gaston Sandra M.
Published: August 2019

Work in models of embryonic development shows that membrane voltage, or resting potential (Vmem), regulates cell behaviors that are disrupted in cancer, including proliferation, migration, apoptosis, homeostasis, and cell-cell signaling. Normal cells and tissues maintain stereotypical patterns of Vmem and these patterns are recognizable in Vmem Imaging as bioelectric signatures. In embryonic tissues, cells that are relatively undifferentiated are usually less negative (depolarized) than fully differentiated cells, and different cell lineages have characteristic bioelectric signatures. Recently, the development of innovative imaging technologies using Voltage Sensitive Dyes (VSDs) allows the visualization of Vmem in living cells and tissues. In culture, Vmem Imaging using VSDs can readily differentiate between sister breast cancer cell lines of low and high metastatic potential (EpH4 and EpH4.2, respectively). Although the cultures are identical in bright field images, Vmem Imaging shows a dramatically more heterogeneous bioelectric signature for EpH4.2 than for EpH4 cells, consistent with reduced gap junctional communication. One of the challenges of Vmem Imaging has been the limited thickness of tissue that can be analyzed using the VSDs (about 50 microns). To overcome this limitation, we have combined innovative Tissue Print technologies with Vmem Imaging in order to visualize patterns of electric potential in living cancer cells obtained from human surgical specimens. Tissue Prints are touch preps that transfer a thin layer of viable cells to a nitrocellulose membrane as an oriented imprint; biomarkers identified on these tissues prints can be mapped back to the source tissue to establish pathology and gene expression correlations. Tissue Print Vmem Imaging of radical prostatectomy specimens shows differentiation of bioelectric signatures between cancer and adjacent benign tissue and between cancer of different grades (Gleason pattern 3 and pattern 4). Such differences in bioelectric signatures represent unexplored cancer phenotypes that are made accessible by Tissue Print Vmem Imaging technologies.

Citation Format: Dany S. Adams, Brian H. Tracey, Larry Takiff, James Kearns, Stephen P. Naber, Sandra M. Gaston. Tissue print Vmem imaging: Visualizing bioelectric signatures in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1933.
Abstract 1936: Label-free optical imaging and characterization of cancer-associated urinary extracellular vesicles: Implications for biomarker discovery
Authors: Barkalifa Ronit , You Sixian , Tu Haohua , Masyr Alison , Kamerer Rebecca , Marjanovic Marina , Park Jaena , Selting Kimberly , Boppart Stephen
Published: August 2019

The discovery of extracellular vesicles (EVs) in all bodily fluids is rapidly increasing the fundamental knowledge of disease mechanisms and the ways in which cells communicate systemically to facilitate tumor progression and metastasis. The type, content, and magnitude of tumor-associated EVs have been linked to tumor invasiveness both in-vitro and in-vivo. In this study we utilized a novel label-free multimodal multiphoton optical imaging method to detect and characterize isolated urinary EVs and classify their optical signatures. Blinded analysis conducted on urinary EVs isolated from 4 healthy dogs and 19 dogs diagnosed with multiple types of cancer showed different optical signatures; cancer-associated urinary EVs were found to have significantly higher NADPH concentrations in comparison to those isolated from healthy dogs. These results suggest a potential label-free optical methodology to detect and characterize EVs by their optical signatures, which can be utilized as possible diagnostic and prognostic biomarkers for cancer.

Citation Format: Ronit Barkalifa, Sixian You, Haohua Tu, Alison Masyr, Rebecca Kamerer, Marina Marjanovic, Jaena Park, Kimberly Selting, Stephen Boppart. Label-free optical imaging and characterization of cancer-associated urinary extracellular vesicles: Implications for biomarker discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1936.
Abstract 1937: pH-responsive tumor-targeted mesoporous silica nanoparticle for the identification of pancreatic cancer using optoacoustic tomography
Authors: MacCuaig William , Samykutty Abhilash , McNally Molly , Nairon Kylie , Banks Surya , Grizzle William , McNally Lacey R.
Published: August 2019

Purpose: Due to inadequate early detection and inability to operate at advanced stages, pancreatic ductal adenocarcinoma (PDAC) has remained one of the most difficult types of cancer to treat. A small range of non-specific symptoms coupled with quick metastasis rate result in a poor 5-year survival rate; 14% for those diagnosed within stage IA, and as low as 1% for those diagnosed during stage IV. Nanoparticles have recently emerged as a potential delivery agent for diagnostic and therapeutic agents, and although clinical success has not been ample due to targeting accuracy issues. This work shows a nanoparticle that has been functionalized with a pancreatic cancer-specific targeting ligand and exhibits specific particle release in pancreatic malignant environment (pH 6.6) as compared to non-malignant environments (pH 7.4). Methods: Wormhole-pored mesoporous silica nanoparticles were formed at 80°C using Tetrapropyl orthosilicate (TPOS) and a scaffold of hexadecyltrimethyl-ammonium bromide (CTAB). Acetic acid and ethanol were used in dialysis procedures to remove the CTAB scaffold and create worm-like pores. Chitosan was added to coat the silica particles and serve as a gatekeeper. IR-780 was added before the solution was acidified to load the dye into the worm-hole particles created. Shortly after, the solution pH was raised back to physiological levels (pH=7.4) to trap the dye within the particle. The particle surfaces were functionalized to attach a targeting ligand pH-low insertion peptide (V7) to conjugate the dye-loaded nanoparticles. Pancreatic adenocarcinoma cells (S2VP10 line) were plated in pH-7.4,6.8, and 6.6 PBS solutions with the loaded particles to assess uptake via near-infrared fluorescence and multispectral optoacoustic imaging. Results: Zeta potential and dynamic light scattering were used to ensure the 63nm size nanoparticle and proper coating. Near-infrared fluorescence imaging showed ~10X increased signal at pH 6.6 as compared to pH=7.4. MSOT imaging ~5X increased signal in the malignant microenvironment environment that is acidic as compared to the non-malignant environment at pH 7.4. Conclusion: The functionalized wormhole mesoporous silica nanoparticles coated with chitosan demonstrated pH-sensitivity in terms of cellular uptake via NIR fluorescence and MSOT imaging.

Citation Format: William MacCuaig, Abhilash Samykutty, Molly McNally, Kylie Nairon, Surya Banks, William Grizzle, Lacey R. McNally. pH-responsive tumor-targeted mesoporous silica nanoparticle for the identification of pancreatic cancer using optoacoustic tomography [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1937.