bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1003: Improving the functional taxonomy of breast cancer-associated fibroblasts by single-cell RNA sequencing reveals clinically relevant cellular subsets
Authors: Bartoschek Michael , Pietras Kristian
Published: August 2018

Aim: The traditional tumor cell-centric view of cancer has been replaced in the past years with the appreciation of the importance of the tumor microenvironment for the malignant phenotype. Cancer-associated fibroblasts (CAFs) are the main constituent of the breast tumor microenvironment, although the exact origin and role of CAF subpopulations in disease initiation, progression and response to treatment remain unclear. Therefore, cancer therapies targeting CAFs are not available.

Methods: Here, we delineate the heterogeneity of CAFs through single-cell RNA sequencing of 768 EPCAM-CD31-CD45-NG2- cells from the MMTV-PyMT mouse model of breast cancer.

Results and discussion: Unbiased transcriptional clustering and differential gene expression analysis revealed four populations, with functionally distinct transcriptional profiles. The existence of these cell populations was further validated by immunostaining in experimental and human tumors revealing a preferential localization of each CAF subtype within the tumor microenvironment. Gene profiles specific to each CAF subpopulation held prognostic capability in clinical cohorts and correlated to known functional gene profiles. Our high-resolution comparison of single-cell transcriptomes in CAFs reveals previously unappreciated functional diversity within the tumor microenvironment.

Conclusions: Ultimately, our delineation of an improved taxonomy of breast CAFs reveals a previously unappreciated functional diversity within the tumor microenvironment and opens the possibility for biomarker-driven development of new drugs for precision medicine.

Citation Format: Michael Bartoschek, Kristian Pietras. Improving the functional taxonomy of breast cancer-associated fibroblasts by single-cell RNA sequencing reveals clinically relevant cellular subsets [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 1003.
Abstract 1004: Tuft cell chemosensory signaling during pancreatic ductal adenocarcinoma
Authors: Hoffman Megan T. , Crawford Howard
Published: August 2018

Pancreatic ductal adenocarcinoma (PDA) is the second most common cause of cancer death in the US with a 10% 5-year survival rate and, as such, is one of the most common deadly cancers. PDA is characterized by a robust stromal reaction, a large portion of which is composed of infiltrating immune cells. This inflammatory response has been previously shown to promote both the initiation and progression of PDA. Current research has been to understand the interaction between tumor cells and infiltrating immune cells and the contribution to cancer progression. In this work we focus on a specific cell population present in the epithelium during pancreatic tumorigenesis called a tuft cell. Tuft cells have been found in luminal surfaces throughout the body, most notably characterized in the intestine where these cells have been shown to directly regulate the immune response to parasitic infection, but have also been found in the nasal, respiratory and bladder epithelium. These solitary chemosensory cells, as they are also known, use a common chemosensory pathway, previously characterized in type-II taste cells, in order to interact with the luminal environment. Interestingly, tuft cells in the metaplastic pancreas uniquely express TRPM5 and alpha-gustducin, both components of the chemosensory cascade, suggesting these cells have the potential to mediate a response to extracellular signals. Pancreatic tuft cells also make an array of cytokines and immunomodulatory factors that can directly alter the immune response in the tumor microenvironment. These data leads us to hypothesize that tuft cells utilize the chemosensory response pathway to remodel the tumor microenvironment in PDA. In order to answer this question, we ablated a key protein in the chemosensory cascade, alpha-gustducin, and found that this loss increases aggressiveness in a model of pancreatitis-associated tumorigenesis. We are currently exploring differences in the tumor microenvironment that may explain these results.

Citation Format: Megan T. Hoffman, Howard Crawford. Tuft cell chemosensory signaling during pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1004.
Abstract 102: RUNX3 inhibits invasiveness and stemness through downregulation of Gli-1 in colon cancer cells
Authors: Kim Bo Ram , Na Yoo Jin , Kim Jung Lim , Jeong Soyeon , Park Seong Hae , Jo Min Jee , Jeong YoonA , Lee Dae-Hee , Oh Sang Cheul
Published: August 2018

Disabled tumor suppressor genes and hyperactive oncogenes contribute considerably to cell fates during cancer development via genetic alterations such as somatic mutations. To date, little is known about how tumor suppressor genes react to diverse oncogenes during tumor progress. Our previous report showed that RUNX3 inhibits metastasis by preventing vascular endothelial growth factor (VEGF) secretion and suppressed endothelial cell growth and tube formation in colorectal cancer. Hedgehog signaling is crucial for the physiological maintenance and self-renewal of stem cells, and its deregulation is responsible for their tumor development. The mechanisms that inhibit this pathway during the proliferation are poorly understood. Here, we show that the tumor suppressor RUNX3 modulates tumorigenesis in response to cancer cells induced by inhibition by oncogene Gli-1 ubiquitination. Moreover, we demonstrated that RUNX3 and Gli-1 expression had inverse correlation in colorectal cancer cells and tissues. We observed a direct interaction between RUNX3 and Gli-1, promoting ubiquitination of Gli-1 at the intracellular level. The increased ubiquitination of Gli-1 was induced through the E3 ligase β-TrCP. This novel RUNX3-dependent regulatory loop may limit the extent and duration of Hedgehog signaling during extension of tumor initiation capacity. Base on this research, a search for agents that can induce RUNX3 might result in new and effective therapies for colorectal cancer.

Citation Format: Bo Ram Kim, Yoo Jin Na, Jung Lim Kim, Soyeon Jeong, Seong Hae Park, Min Jee Jo, YoonA Jeong, Dae-Hee Lee, Sang Cheul Oh. RUNX3 inhibits invasiveness and stemness through downregulation of Gli-1 in colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 102.
Abstract 1028: Patient-derived xenografts undergo mouse-specific tumor evolution
Authors: Ben-David Uri , Ha Gavin , Tseng Yuen-Yi , Greenwald Noah F. , Oh Coyin , Shih Juliann , McFarland James M. , Wong Bang , Boehm Jesse S. , Beroukhim Rameen , Golub Todd R.
Published: August 2018

Patient-derived xenografts (PDXs) have become a prominent cancer model system, as they are presumed to faithfully represent the genomic features of primary tumors. Here we monitored the dynamics of copy number alterations (CNAs) in 1,110 PDX samples across 24 cancer types. We observed rapid accumulation of CNAs during PDX passaging, often due to selection of pre-existing minor clones. CNA acquisition in PDXs was correlated with the tissue-specific levels of aneuploidy and genetic heterogeneity observed in primary tumors. However, the particular CNAs acquired during PDX passaging differed from those acquired during tumor evolution in patients. Several CNAs recurrently observed in primary tumors gradually disappeared in PDXs, indicating that events undergoing positive selection in humans can become dispensable during propagation in mice. Importantly, the genomic stability of PDXs was associated with their response to chemotherapy and targeted drugs. These findings have important implications for PDX-based modeling of human cancer.

Citation Format: Uri Ben-David, Gavin Ha, Yuen-Yi Tseng, Noah F. Greenwald, Coyin Oh, Juliann Shih, James M. McFarland, Bang Wong, Jesse S. Boehm, Rameen Beroukhim, Todd R. Golub. Patient-derived xenografts undergo mouse-specific tumor evolution [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 1028.
Abstract 103: Expression and regulation of MMP9 and RECK in human oral squamous cell carcinoma progression and invasion
Authors: Pramanik Kamdeo Kumar , Rana Basabi , Rana Ajay , Mishra Rajakishore
Published: August 2018

Purpose: Matrix metalloproteinase-9 (MMP-9), a Zn-dependent endopeptidase is known to play a central role in oral cancer pathogenesis. Recently we observed differential function of MMP-9 and reversion-inducing cysteine-rich protein with Kazal motifs (RECK) respectively in OSCC invasion. We hypothesize that the downregulation of MMP-9 via RECK promotes human oral cancer progression and invasion.

Experimental procedure: The mRNA expression, protein expression and activity of MMP-9/2, expression of transcription factors like Snail, c-Myc, β-catenin and protein-protein interaction of TIMP1/RECK with MMP9 and promoter methylation of RECK gene were assessed by using RT-PCR, immunohistochemistry (IHC), Western blotting, co-immunoprecipitation, gelatin zymography, and bisulphate modification-PCR analyses. Wnt5a and LPA mediated MMP-9 regulation as well as the MMP-9 regulation by the exogenously expression of RECK, GSK3β (WT) or non phosphoryable active GSK3β (S9A) was also performed in oral tongue SCC derived SCC-9 cells.

Summary: We observed up-regulation/activation of MMP-9 was associated with decreased expression of RECK and progression and invasion of OSCC. MMP-9 expression/ activity was positively correlated with inactivation of GSK3β signaling. Conversely, a link between inactivation of GSK3 signaling and the progressive silencing of RECK gene through promoter hypermethylation was observed. Finally, Wnt5a and LPA mediated increased MMP-9 and decreased RECK expressions were observed.

Conclusion: Taken together, these results demonstrated the influence of a novel signaling axis GSK3β-RECK-MMP-9 to drive OSCC progression and invasion. Thus, targeting this pathway may be exploited for treating OSCC.

Citation Format: Kamdeo Kumar Pramanik, Basabi Rana, Ajay Rana, Rajakishore Mishra. Expression and regulation of MMP9 and RECK in human oral squamous cell carcinoma progression and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 103.
Abstract 1034: Genotyping of patient-derived xenograft tumors using short tandem repeats: Challenges and observations in ensuring concordance across passages for effective preclinical studies
Authors: Sisson Bridgette , Hesse Andrew , Soucy Melissa , Bergeron Daniel , Burns Shelbi , Kelly Kevin , Jocoy Emily , Bundy Margaret , Reddi Honey V.
Published: August 2018

Patient derived xenograft (PDX) tumor models represent the closest step to in-human trials for cancer drug research and are therefore an invaluable resource. Implantation of a cancerous tissue from a patient's primary tumor directly into an immune-deficient mouse, generating a xenograft tumor potentially mimics the tumor microenvironment in the patient and allows for the evaluation of therapies specific to a particular tumor. One of the significant challenges associated with PDX tumors is the potential loss of the human component or the take-over by mouse tissue with every passage into a new mouse model. Short tandem repeats (STRs) are short intronic sequences of DNA that are highly polymorphic and contain a pattern of nucleotides repeated in tandem. STRs can be found in distinct regions of both prokaryotes and eukaryotes organisms, with each organism containing a unique combination of the STR allelic frequencies that allow for individuals to be unambiguously identified. STR analysis is routinely used to genotype PDX tumors comparative to the original resected human cancerous tissue, ensuring concordance with original tissue and to identify mouse tissue contamination with progressive passages. The Jackson Laboratory for Genomic Medicine's CLIA laboratory has evaluated over 170 PDX tumor samples from multiple mouse model passages across 105 diverse PDX mouse models. Of those samples analyzed, genomic DNA for 33 had been extracted from fresh frozen tissue and for 143 extracted from Formalin Fixed Paraffin embedded tissue (FFPE). The current study presents the challenges and discrepancies observed in our evaluation relative to number of passages for each tumor, sample type (FFPE vs fresh frozen) - reflective of tumor heterogeneity and also potential encroachment by mouse tissue. We also present results of a cross institutional comparative STR analysis study in 123 samples wherein discordance observed in certain cases were a result of differing sample types, suggestive that tumor heterogeneity can affect the STR results and could be a leading cause for cross institutional discordant genotyping results.

Citation Format: Bridgette Sisson, Andrew Hesse, Melissa Soucy, Daniel Bergeron, Shelbi Burns, Kevin Kelly, Emily Jocoy, Margaret Bundy, Honey V. Reddi. Genotyping of patient-derived xenograft tumors using short tandem repeats: Challenges and observations in ensuring concordance across passages for effective preclinical studies [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 1034.
Abstract 1043: Patient-derived and cell line xenograft growth in the B6;129-Rag2
tm1Fwa
IL2rg
tm1Rsky/DwlHsd (R2G2) mouse model
Authors: Naden Jamie L.
Published: August 2018

Immunodeficient mouse models are helping to advance the field of oncology. A new model on the market, the B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) knockout mouse, lacks responsiveness to common gamma chain cytokines, including IL-2, IL-4, IL-7, IL-9 and IL-15. In addition, this model exhibits defects in lymphoid development and so lacks mature lymphocytes of the B, T, and natural killer (NK) cell lineages. Herein we describe growth of multiple patient-derived (PDX) and tumor cell line xeno- (CDX) and allografts in the R2G2 immunodeficient mouse model. The PDXs examined include colorectal and head and neck cancers. The CDXs studied include esophageal (OE33 and FLO1) and stomach cancer (AGS). Tumor growth data were also collected from two allografts of murine colorectal cancer (CT26) and B-cell lymphoma (A20) cells. Colorectal PDX tissue was subcutaneously implanted bilaterally into five male R2G2 and five male NSG mice. Growth was comparable between the R2G2 and the NSG mouse models; however, the standard error was much lower in the R2G2 strain. Head and neck PDX 626 and 635 was transplanted in 2.2 mm2 tissues into 4 sections of each of 2 R2G2 mice each (n=2/PDX), and 100% of mice developed either one or two tumors. The human esophageal adenocarcinoma OE33 cells were implanted into the left and right flanks of three each of R2G2, athymic nude and SCID mice. There was a 100% take rate in R2G2 mice, 0% in SCID mice and 17% in athymic nude mice. The human esophageal adenocarcinoma FLO1 cells were examined in two studies. In both studies, cells were injected into both flanks of R2G2 and SCID mice. Study A also examined growth in athymic nude mice. In study A, no tumor growth was seen in athymic nude or SCID mice, whereas the take rate was 100% in R2G2 mice. In study B, the take rate was 100% in both the R2G2 and the SCID mice, although differences were seen in growth rate. Human gastric adenocarcinoma AGS cells were implanted in both flanks of four each of R2G2 and SCID mice. The take rate was 75% in R2G2 mice and 0% in SCID mice. Head and neck squamous cell carcinoma SQ20b cells were implanted in twenty R2G2 mice and take rate was 90%. Growth of two allogeneic tumor lines was also examined. The mouse colon carcinoma CT26 cells were implanted in ten R2G2 mice and took in 100% of the mice. Mouse B-cell lymphoma A20 cells were implanted in ten R2G2 mice and take rate was 100%. Both allografts grew to 1000mm3 by 13 days post implantation. These data provide evidence that the R2G2 mouse model is a valuable tool for oncology programs including cell line tumor models research, with high take rates and quick growth of allogeneic models.

Citation Format: Jamie L. Naden. Patient-derived and cell line xenograft growth in the B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) mouse model [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 1043.
Abstract 1044: Establishment and characterization of rare breast patient-derived xenograft models as a potential resource for personalized medicine
Authors: Kuasne Hellen , Savage Paul , Ramirez Constanza Martinez , Liu Leah , Muñoz-Ramos Valentina , Pilon Virginie , Monast Anie , Johnson Radia , Bertos Nicholas , Asselah Jamil , Bouganim Nathaniel , Petrecca Kevin , Meterissian Sarkis , Omeroglu Atilla , Basik Mark , Park Morag
Published: August 2018

Breast cancer (BC) exhibits a wide range of morphologic phenotypes and gene expression profiles. Most of the studies that led to the identification of intrinsic molecular subtypes in BC were limited to invasive ductal carcinomas of the breast and did not take rare histopathologic subtypes into account. Rare histopathologic BC subtypes (collectively less than 2% of all breast cancer) have particular prognostic and clinical characteristics. There is no current established treatment that takes into account the specificity of rare BC subtypes. This is mainly due to the absence of clinical trials to determine the optimal management of these rare pathologies. The establishment of relevant preclinical models and molecular characterization of rare BC subtypes is essential for identifying directed and suitable therapeutic regimens for BC patients diagnosed with these rare histopathologic variants. Patient-derived xenograft (PDX) has been recognized as a valuable method to evaluate the clinical diversity of breast cancer. These models were shown to be predictive of clinical outcomes and are being used for preclinical drug evaluation, biomarker identification, and personalized medicine strategies. We developed a cohort of eight BC rare histopathologic subtypes, including four metaplastic, one adenoid cystic, one IDC pleomorphic, one neuroendocrine, and one mucinous BC subtype. These PDXs and their primary tumors were submitted to whole-genome sequencing (WGS) and RNA sequencing. We also evaluated a total of 255 proteins by reverse phase protein array (RPPA) in these PDX samples. We are currently performing conditional reprogramming experiments to generate cell lines from these rare BC PDXs. Our preliminary results indicate that pathways related to PI3K/AKT, ERK/MAPK, mTOR, HGF, ERBB, AMPK and IL3 signaling are disrupted in rare BC subtypes. Several genes belonging to these pathways are dysregulated in rare BC tumors, and therefore represent potential therapeutic targets for personalized treatment.

Citation Format: Hellen Kuasne, Paul Savage, Constanza Martinez Ramirez, Leah Liu, Valentina Muñoz-Ramos, Virginie Pilon, Anie Monast, Radia Johnson, Nicholas Bertos, Jamil Asselah, Nathaniel Bouganim, Kevin Petrecca, Sarkis Meterissian, Atilla Omeroglu, Mark Basik, Morag Park. Establishment and characterization of rare breast patient-derived xenograft models as a potential resource for personalized medicine [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 1044.
Abstract 1053: Highly characterized patient-derived PDX breast cancer collection for preclinical efficacy studies
Authors: Melton Michele
Published: August 2018

We have acquired a world-class collection of highly characterized patient-derived breast cancer models (PDX) from Washington University, St. Louis, MO (1). We have established growth curves for 17 of these lines, 6 ER+, 2 HER2+ and 4 triple-negative (TNBC) models beyond what was originally published. Continued characterization of these models is ongoing in our lab to ensure the integrity of the models is being maintained over multiple rounds of propagation. Short tandem repeat (STR) analysis, sequencing, quantitative RTPCR and Western blots were run to confirm genotypic and phenotypic fidelity of many of these model. To further characterize these models, RNA samples have been run using NanoString PanCancer gene expression analysis with 770 genes from 13 cancer-associated pathways including: MAPK, STAT, PI3K, RAS, Cell Cycle, Apoptosis, Hedgehog, Wnt, DNA Damage Control, transcription regulation, chromatin modification and TGF-β. Results: These data sets show that we have maintained the integrity of the WHIM PDX models over several passages in mice and provided confidence in the use of these models for preclinical efficacy studies.

Reference: 1. Li et al., Cell Reports 2013;4:1116-30.

Citation Format: Michele Melton. Highly characterized patient-derived PDX breast cancer collection for preclinical efficacy studies [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 1053.
Abstract 1055: Lymphotoxin-alpha plays key roles in lymphoproliferative lesions arising in patient-derived cancer xenografts
Authors: Chijiwa Tsuyoshi , Komura Daisuke , Haraguchi Mizuha , Hashimoto Haruo , Suemizu Hiroshi , Katayama Makoto , Nakamura Yoshiyasu , Furukawa Daisuke , Isagawa Takayuki , Katoh Hiroto , Moriya Takashi , Ishikawa Shumpei , Miyagi Yohei , Nakamura Masato
Published: August 2018

Patient-derived xenograft (PDX) mouse models from engrafting fresh surgical specimens directly onto immunodeficient mice have enabled the development of more relevant in vivo models for human cancers. PDX models retain similar morphology, heterogeneities, and molecular signatures with the original cancers. Moreover, PDXs with clinical information, termed “cancer xenopatients”, are remarkable systems in personalized medicine for cancer. During the establishment process, a transplantable lymphoproliferative lesion (LPL) that replaces the original tumor cells sometimes occurs in severely immunodeficient mice. Although the occurrence of LPL is the most problematic aspect of the establishment of PDX, its mechanisms are unknown. We previously reported that the LPL phenomenon resembled the graft-versus-host disease (GVHD)-like mechanism and was suppressed by anti-GVHD agents. In the present study, we genomically analyzed the detailed mechanism of LPL in PDX mouse models. In our previous study, we established three LPL-PDX/NOG lines. These LPL-PDX lines were re-transplanted onto NOG mice and the xenograft tumors were then harvested for study. Genome sequencing and comprehensive analyses of tumor-stroma interactions (CAncer-STromal INteractome analysis, CASTIN) were performed on mRNA of these LPL-PDX/NOGs. Immunohistochemistry demonstrated that LPL tissues consisted of B-cells, T-cells, and differentiated plasma cells. CASTIN revealed tumor-stroma interactions in PDX/NOG comprehensively and quantitatively at the gene expression level by distinguishing gene arrangements in human tissue (cancer or LPL) from mouse tissue (stroma) by signal strength and dependency. Some strong interactions were found for these LPL-PDX/NOGs; VEGFB-FLT1, LTA-LTBR, LTA-TNFRSF1A, IL13-IL4R, IL13-IL13RA1, and SEMA4F-NRP2. Suppression of LPL-PDX/NOG by the anti-GVHD agent FK506 (1.0 mg/kg/day, 4 weeks) decreased interactions of LTA-LTBR and LTA-TNFRSF1A. Based on these results, LPLs during establishment of PDX occur due to a lymphotoxin-alpha-related mechanism. Controlling lymphotoxin-alpha related interactions may improve the establishment rate of PDX in severely immunodeficient mice.

Citation Format: Tsuyoshi Chijiwa, Daisuke Komura, Mizuha Haraguchi, Haruo Hashimoto, Hiroshi Suemizu, Makoto Katayama, Yoshiyasu Nakamura, Daisuke Furukawa, Takayuki Isagawa, Hiroto Katoh, Takashi Moriya, Shumpei Ishikawa, Yohei Miyagi, Masato Nakamura. Lymphotoxin-alpha plays key roles in lymphoproliferative lesions arising in patient-derived cancer xenografts [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 1055.
Abstract 1060: Longitudinal profiling of plasma derived extracellular vesicles (EVs) from women presenting with metastatic triple-negative breast cancer (mTNBC) informs on metastatic location and treatment outcome
Authors: Lindsay Sinéad , McIntyre Jennifer , Gubbins Luke , Smith Martina , Kerr Laura , Kavanagh Emma , Higgins Michaela J. , Hennelly Bryan , Dowling Paul , Byrne Hugh , McCann Amanda
Published: August 2018

Background: Women presenting with Triple Negative Breast Cancer (TNBC) have an increased risk of recurring at distant visceral metastatic sites such as brain, lungs and liver rather than bone compared to oestrogen-receptor-positive breast cancer. (Rakha et al., 2011, Wood 2004). The preferential homing of TNBC cancer cells to these sites is not clearly understood, but is increasingly being attributed to the ability of extracellular vesicles (EVs) released by chemoresistant cancer cells to circulate to distant sites creating metastatic niches and paving the way for organ-specific metastases.

Hypothesis: We hypothesise that the physical EV properties, macromolecular and proteomic content of these EVs could potentially determine the risk of metastasis and align with these “preferred” metastatic locations.

Methodology: This project uses nanoparticle tracking analysis (NTA), infra-red and mass spectroscopy to generate unique patient-specific EV longitudinal signatures of women diagnosed with organ defined metastatic TNBC. These signatures hold potential as “unique identifiers” of clinically documented organ-specific metastases. All profiles generated are compared longitudinally to the patient's initial baseline sample and also to age matched female control plasma samples.

Results and Conclusions: Preliminary results have developed SOPs to (i) successfully enrich for EVs from patient plasma samples and (ii) employ nanoparticle tracking analysis, infra-red and mass spectroscopic profiling to develop unique longitudinal patient specific EV signatures. The longitudinal profiling of EV signatures from patients with metastatic TNBC will potentially (a) predict response to therapy, (b) uniquely identify risk of and potential location of metastatic site/sites and (c) inform on patient outcome. Following this multidisciplinary profiling of our mTNBC, our remit is to recapitulate this profiling into the primary TNBC setting to identify potential sites of organ specific metastases ahead of any detection on an x-ray or scan, thereby informing on clinical management.

Citation Format: Sinéad Lindsay, Jennifer McIntyre, Luke Gubbins, Martina Smith, Laura Kerr, Emma Kavanagh, Michaela J. Higgins, Bryan Hennelly, Paul Dowling, Hugh Byrne, Amanda McCann. Longitudinal profiling of plasma derived extracellular vesicles (EVs) from women presenting with metastatic triple-negative breast cancer (mTNBC) informs on metastatic location and treatment outcome [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 1060.
Abstract 1061: Advanced quantitative mass-spectrometry-based SILAC proteome profiling of Y-box binding protein-1 (YB-1) overexpressing breast cancer cells unravels proteins involved in metastasis
Authors: Lim Jia Pei , Nair Sunitha , Shyamasundar Sukanya , Gunaratne Jayantha , Bay Boon Huat
Published: August 2018

Metastasis is responsible for the majority of cancer related deaths and remains a global medical challenge in cancer treatment. The Y-box binding protein-1 (YB-1), a DNA and RNA binding protein, is known to regulate a plethora of fundamental cellular processes, including cell proliferation, chemoresistance and DNA repair. There is also cumulative evidence that YB-1 enhances breast cancer spread although the exact mechanism of YB-1-mediated metastasis is still not clear. Hence, we aim to determine the role of YB-1 overexpression in breast cancer progression. We first stably overexpressed YB-1 in non-aggressive MCF7 breast cancer cells (MCF7-YB-1) using a turboGFP tagged plasmid and performed phenotypic assays. We observed a significant increase in cell migration in MCF7-YB-1 in the Boyden chamber migration assay, which was substantiated by performing live imaging of the cells in a wound healing assay. Furthermore, a significant decrease in cell proliferation with G1 phase arrest was observed by the alamarBlue assay and flow cytometry analysis of propidium iodide staining, respectively for MCF7-YB-1 cells. We then performed a whole proteome analysis by stable isotope labelling with amino acids in cell culture (SILAC), a powerful proteomics platform. The whole proteome analysis identified 122 upregulated proteins and 120 downregulated proteins in the MCF-7-YB-1 cells. Bioinformatics analysis of the differentially regulated proteins using the Metacore software revealed the involvement of proteins that promote cell migration and invasion, such as those belonging to the S100 family which are enriched in the extracellular vesicles and exosome compartment, providing some mechanistic insights into the metastasis promoting role of YB-1. Taken together, our results showed that YB-1 could enhance the migratory potential of breast cancer cells, an important step in the metastatic cascade and therefore warrants a more in-depth study.

Acknowledgement: Jia Pei Lim is a recipient of Ong Hin Tiang Scholarship in Cancer Research. This research was supported by Ministry of Education Grant (MOE2013-T2-1-129).

Citation Format: Jia Pei Lim, Sunitha Nair, Sukanya Shyamasundar, Jayantha Gunaratne, Boon Huat Bay. Advanced quantitative mass-spectrometry-based SILAC proteome profiling of Y-box binding protein-1 (YB-1) overexpressing breast cancer cells unravels proteins involved in metastasis [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 1061.
Abstract 1074: Expression of p39 and Rb in lung cancer and its correlation with clinicopathologic parameters
Authors: Perez-Morales Jaileene , Rivera Stephanie , Torres Bryan , Rodriguez Xavier , Isidro Angel , Santiago-Cardona Pedro
Published: August 2018

Lung cancer is characterized by its aggressiveness and proclivity for early metastasis. A predictor of metastasis of NSCLC rely on post-resection evaluation of tumor histology, which is a severe limitation since only 15% of the patients are diagnosed with resectable disease. Hence, there is a need to characterize biomarkers with metastasis-predicting value in pre-resection small biopsy specimens. Rb is a tumor suppressor inactivated due to hyper-phosphorylation in NSCLC. Our preliminary data relates the Rb S249 phosphorylation to an epithelial-to-mesenchymal transition (EMT), a trait strongly associated to metastasis and increase in CDK5 activator p39. Here, we examined the prognostic significance of Rb S249 and p39 as biomarkers of metastasis staging in non-small cell lung cancer. Our hypothesis is that Rb hyper-phosphorylation in S249 can have prognostic value by being associated with a metastatic phenotype and EMT. To determine p39 and Rb phosphorylation in lung cancer, immunohistochemistry analysis was performed on two lung cancer microarrays. Three pathologists observed the microarrays and scored them using the all-red scoring system. P39 and Rb phosphorylation in S249 were more present in lung tumors than adjacent normal tissue. P39 positive expression had a positive correlation with staging, lymph node involvement, and metastasis. Rb phosphorylation in residue S249 had a positive correlation with histological grading. Using general linear model, we determined that staging can be predicted when combining phosphorylation of Rb S249 and p39. Our analysis suggests that p39 and Rb S249 are likely to be diagnostic biomarkers for lung cancer metastasis.

Citation Format: Jaileene Perez-Morales, Stephanie Rivera, Bryan Torres, Xavier Rodriguez, Angel Isidro, Pedro Santiago-Cardona. Expression of p39 and Rb in lung cancer and its correlation with clinicopathologic parameters [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 1074.
Abstract 1084: TRAF4-mediated NGF receptor TrkA ubiquitination regulates prostate cancer metastasis
Authors: Singh Ramesh , Karri Dileep , Shao Jiangyong , Dasgupta Subhamoy , Huang Shixia , Edwards Dean P. , O'Malley Bert W. , Yi Ping
Published: August 2018

Receptor tyrosine kinases (RTKs) are important drivers of cancers. In addition to genomic alterations, aberrant activation of wild type RTKs play an important role in driving cancer progression. However, the underlying mechanisms of how RTKs drive prostate cancer remains incompletely characterized. Here we show that non-proteolytic ubiquitination of RTK regulates its kinase activity and contributes to RTK-mediated prostate cancer metastasis. TRAF4, a RING domain E3 ubiquitin ligase, is highly expressed in metastatic prostate cancer. We demonstrated here that it is a key player in regulating RTK mediated prostate cancer metastasis. We further identified TrkA, a neurotrophin receptor tyrosine kinase, as a TRAF4-targeted ubiquitination substrate that promotes cancer cell invasion and genetic ablation of TrkA abolished TRAF4-dependent cell invasion. TRAF4 promoted atypical K27 and K29-linked ubiquitination at the TrkA kinase domain and increased its kinase activity. Mutation of TRAF4-targeted ubiquitination sites abolished TrkA tyrosine auto-phosphorylation and its interaction with downstream effector proteins. TRAF4 knockdown also suppressed nerve growth factor-stimulated TrkA downstream p38 MAPK activation and subsequent invasion-associated gene expression. Furthermore, elevated TRAF4 levels significantly correlated with increased NGF-stimulated invasion-associated gene expression in prostate cancer patients, indicating this signaling axis is significantly activated during oncogenesis. Our results revealed a post-translational modification mechanism contributing to aberrant non-mutated receptor tyrosine kinase activation in cancer cells.

Citation Format: Ramesh Singh, Dileep Karri, Jiangyong Shao, Subhamoy Dasgupta, Shixia Huang, Dean P. Edwards, Bert W. O'Malley, Ping Yi. TRAF4-mediated NGF receptor TrkA ubiquitination regulates prostate cancer metastasis [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 1084.
Abstract 1088: Defining bone metastatic potential of Abi1 CRISPR prostate cancer cell lines
Authors: Nappi Alexander , Post Dawn , Das Disharee , Joshi Ishita , Oest Megan , Bratslavsky Gennady , Kotula Leszek
Published: August 2018

Prostate cancer is the second most common cancer in American men, and it is the third leading cause of cancer-related deaths in American men. Distant stage and metastatic disease is often treated with androgen-deprivation therapy (ADT); however, this treatment ultimately fails, leading to castration-resistant prostate cancer (CRPC). This further emphasizes the need for better therapeutic targets. Our work focuses on understanding the role of the Abi1 gene in prostate cancer tumor progression. Our current in vitro data demonstrate an invasive phenotype with our CRISPR-mediated Abi1 knockout (KO) cell lines. In total, using CRISPR-mediated technology we have generated four Abi1 gene KO cell lines, PC-3, DU145, LnCaP, and RWPE-1. Importantly, the androgen-dependent cell line, LNCaP, demonstrated upregulation of Abi1 to the androgen receptor stimulation and the opposite response to the receptor inhibition upon treatment with enzalutamide. These data indicated that low Abi1 level could be induced by ADT therapies and are consistent with our previous observation of low Abi1/WAVE complex in prostate tumor samples. To learn more about this mechanism, we set out to investigate the role of Abi1 in castrated mice. The overall goal of the experiment is to compare the ability of cells lacking the Abi1 gene to engraft in bone under androgen deprivation condition and compare it to the control cells and examine any change in the engraftment phenotype. It is hoped that this research will lead to better understanding of the Abi1/WAVE complex role in CRPC.

Supported by NCI R01 CA16101.

Citation Format: Alexander Nappi, Dawn Post, Disharee Das, Ishita Joshi, Megan Oest, Gennady Bratslavsky, Leszek Kotula. Defining bone metastatic potential of Abi1 CRISPR prostate cancer cell lines [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 1088.
Abstract 1090: The role of connexin43 in prostate cancer motility
Authors: Aloliqi Abdulaziz A.
Published: August 2018

Prostate cancer (PC) is one of the most common male cancers in United States. PC lethality is due to metastatic growth, when cancer cells move from the primary tumor site to a secondary site. A key step in metastsis is the acquisition of motility. Connexins (Cx) play a major role in cell-cell communication and growing evidence demonstrates their contribution to cell motility. Cx43 is up-regulated in cultured PC3 metastatic prostate cancer cells. To understand the role of Cx43 in migration we asked whether silencing Cx43 would decrease PC3 cell migration. Using cells in which short interfering RNA (sh-RNA) silenced Cx43 mRNA, we showed that silencing Cx43 decreased motility of sh-Cx43 PC3 cells in Boyden chamber assays. To confirm these results in another biological system, we performed cellular wound healing assays. Silencing Cx43 decreased motility of PC3 cells reducing movement to wounded area, leaving more area open in the Cx43 inhibited cell lines. The wound healing assay showed that knockdown Cx43 altered directional movement, velocity and distance. These results supported those of our Boyden assays showing that loss of Cx43 decreases the migratory ability of PC3. Conversely, the overexpression of Cx43 in LNCaP cells, non-migratory prostate cancer cells that lack Cx43 expression, increased their motility. A comparison of cell lines engineered to overexpress or to silence Cx43 confirms the importance of Cx43 in the motility of prostate cancer cells in vitro. Although the mechanism whereby Cx43 regulates motility is still unclear, our preliminary data revealed that Cx43 co-immunoprecipitated with N-cadherin, suggesting that they form a complex in PC3 cells. Cell fractionation studies of PC3 control cells showed that N-cadherin was located in the cell membrane and not detected free in the cytoplasm, but in sh-Cx43 PC3 cells that lack Cx43, the distribution of N-cadherin protein was altered and significant levels of N-cadherin were also detected in cytoplasmic fractions. Whether changes in N-cadherin distribution is a direct or indirect result of loss of Cx43 in sh-Cx43 PC3 cells, is unclear. However, modulation of Cx43 levels in prostate cancer cells does alter motility and N-cadherin distribution. These results indicate that Cx43 and N-cadherin are in close association in PC3 cells and their interaction may play an important role in prostate cancer cell motility. A better understanding of the requirements of migratory PC cells may lead to more effective targeting of key steps in PC progression to lethal metastatic disease.

Citation Format: Abdulaziz A. Aloliqi. The role of connexin43 in prostate cancer motility [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 1090.
Abstract 1091: Role of growth differentiation factor-15 in prostate cancer-bone environment: Understanding on bone metastasis
Authors: Siddiqui Jawed A. , Muniyan Sakthivel , Seshacharyulu Parthasarathy , Rachagani Satyanarayana , Gupta Suprit , Nasser Mohd W. , Datta Kaustubh , Batra Surinder K.
Published: August 2018

Background: Prostate cancer (PCa) cells preferentially metastasize to bone and skeleton resulting in the poor 5-year survival of less than 1%. Growth differentiation factor-15 (GDF15) is overexpressed in several cancers, including PCa. However, the role of GDF15 in prostate cancer bone metastasis remains elusive. The interaction between cancer cells and the bone microenvironment forms a vicious cycle that increases bone deformities and drives tumor growth in the bone. Therefore, we question whether GDF15 secreted by the PCa cells alters the bone environment by uncoupling bone formation and resorption. Methods: Murine calvarial-derived osteoblast (MCO) cells were used for the functional effect of GDF15 on differentiation, proliferation, and mineralization. Mineralization of mouse bone marrow stromal cell (BMSCs) derived osteoblasts was determined by Alizarin Red S staining. Bone marrow macrophages (BMMs) were isolated from C57 mice and cultured along with M-CSF and RANKL to identify osteoclasts. Results: Conditioned media (CM) from PCa (LNCaP and C4-2B) cells and rhGDF15 increased osteoblast proliferation and differentiation in MCO cells. However, GDF15 deletion in PCa (LNCaP and C4-2B) cells prevented PCa-mediated osteoblast differentiation. Further, CM from PCa and rhGDF15 increased the alkaline phosphatase+ CFU-F (Colony Forming Unit- Fibroblast) in mouse bone marrow stromal cells culture. Supportively, stable knockout of GDF15 decreased mineralized nodule formation in MCO. qRT-PCR assay revealed that the PCa and rhGDF15 increased mRNA expression of osteoblast-related genes such as alkaline phosphatase, Runx-2, collagen type-1 and osteocalcin. PCa cells derived CM and rhGDF15 increase the osteoblast-derived osteoclastic signal (RANKL/OPG ratio), suggesting that GDF15 promotes RANKL-dependent osteoclastogenesis. Furthermore, rhGDF15 treatment increased TRAP-positive multinucleated cells, pit resorption and osteoclast-specific genes such as TRAP, Cathepsin K, Carbonic anhydrase and NFATc1 in BMMs. Conclusions: Altogether, our results suggest that GDF15 is crucial for PCa-mediated osteoblast differentiation and mineralization. GDF15 also increased bone marrow osteoclast formation and its function. Our finding suggests that GDF15 mediated increase in osteoblastic-RANKL further enhance osteoclast activity and favor PCa cell to metastasize to bone.

Citation Format: Jawed A. Siddiqui, Sakthivel Muniyan, Parthasarathy Seshacharyulu, Satyanarayana Rachagani, Suprit Gupta, Mohd W. Nasser, Kaustubh Datta, Surinder K. Batra. Role of growth differentiation factor-15 in prostate cancer-bone environment: Understanding on bone metastasis [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 1091.
Abstract 1093: Epithelial-to-mesenchymal transition conveys resistance to restrictive growth conditions through increased autophagic flux
Authors: Zhao Lei , Nauseef Jones , Buchakjian Marisa , Barnes Matthew , Henry Michael
Published: August 2018

Tumor progression requires cancer cells to adapt to varying microenvironments both in the primary tumor and in metastatic end organs. Metastasis is enhanced by epithelial-to-mesenchymal transition (EMT), which facilitates invasiveness and confers resistance to local stresses, such as hypoxia and nutrient limitation, although EMT is not necessarily required for these processes. Among these stressors, growth-factor (GF) deprivation may induce or select for cells that bear the hallmarks of EMT (EMT-like cells). The mechanisms by which EMT-like cells survive GF deprivation (restrictive growth conditions), which may occur when metastatic cancer cells are removed from GFs resident in the primary tumor microenvironment, remain largely uncertain. Herein we show that EMT confers upon cells the ability to maintain proliferative potential, following extended serum deprivation. We found that EMT-like prostate cancer cells (TEM 4-18) demonstrate elevated survival and reduced senescence compared to their more epithelial counterparts (PC-3E) in both short- and long-term exposure to serum withdrawal. In addition, resistance to anchorage-dependent growth-induced apoptosis (Anoikis) due to elevated autophagy is also evident in EMT-like cells. Forced expression of snail1 conferred survival advantages to PC-3 cells. Conversely, CRISPR-mediated knockout of Zeb1 reversed EMT status in TEM4-18 cells and survival advantage in restrictive growth conditions. We found that EMT-like cells exhibit elevated autophagic flux. Genetic interference with autophagy in EMT-like cells reversed their survival advantage and increased senescence in restrictive growth conditions as well as anoikis resistance. Our results suggest that the metastatic capabilities of EMT-like cells depend not only on their established invasive characteristics, but also on increased autophagy tied to the EMT-like state, and its role in surviving restrictive growth conditions.

Citation Format: Lei Zhao, Jones Nauseef, Marisa Buchakjian, Matthew Barnes, Michael Henry. Epithelial-to-mesenchymal transition conveys resistance to restrictive growth conditions through increased autophagic flux [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 1093.