Laurie Ailles

A role for Wnt signalling in self-renewal of haematopoietic stem cells

Haematopoietic stem cells (HSCs) have the ability to renew themselves and to give rise to all lineages of the blood; however, the signals that regulate HSC self-renewal remain unclear. Here we show that the Wnt signalling pathway has an important role in this process. Overexpression of activated β-catenin expands the pool of HSCs in long-term cultures by both phenotype and function. Furthermore, HSCs in their normal microenvironment activate a LEF-1/TCF reporter, which indicates that HCSs respond to Wnt signalling in vivo. To demonstrate the physiological significance of this pathway for HSC proliferation we show that the ectopic expression of axin or a frizzled ligand-binding domain, inhibitors of the Wnt signalling pathway, leads to inhibition of HSC growth in vitro and reduced reconstitution in vivo. Furthermore, activation of Wnt signalling in HSCs induces increased expression of HoxB4 and Notch1, genes previously implicated in self-renewal of HSCs. We conclude that the Wnt signalling pathway is critical for normal HSC homeostasis in vitro and in vivo, and provide insight into a potential molecular hierarchy of regulation of HSC development.

Frequency of cells expressing CD44, a Head and Neck cancer stem cell marker: Correlation with tumor aggressiveness

We previously identified by flow cytometry a Lineage‐CD44+ (Lin‐CD44+) subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma (HNSCC). We now correlate clinical and histologic factors with Lin‐CD44+ cell frequency.

Separating Stem Cells by Flow Cytometry: Reducing Variability for Solid Tissues

Until there are valid identifiers that visualize stem cells in vivo, we rely upon flow cytometry to enrich for subpopulations with stem cell function. However, data reporting styles for flow cytometric analyses are typically inconsistent, creating challenges in comparing results across publications. In our view, clear reporting guidelines could improve reproducibility of stem cell analyses in solid tissues.

Oncolytic targeting of renal cell carcinoma via encephalomyocarditis virus.

Apoptosis is a fundamental host defence mechanism against invading microbes. Inactivation of NF‐κB attenuates encephalomyocarditis virus (EMCV) virulence by triggering rapid apoptosis of infected cells, thereby pre‐emptively limiting viral replication. Recent evidence has shown that hypoxia‐inducible factor (HIF) increases NF‐κB‐mediated anti‐apoptotic response in clear‐cell renal cell carcinoma (CCRCC) that commonly exhibit hyperactivation of HIF due to the loss of its principal negative regulator, von Hippel–Lindau (VHL) tumour suppressor protein. Here, we show that EMCV challenge induces a strong NF‐κB‐dependent gene expression profile concomitant with a lack of interferon‐mediated anti‐viral response in VHL‐null CCRCC, and that multiple established CCRCC cell lines, as well as early‐passage primary CCRCC cultured cells, are acutely susceptible to EMCV replication and virulence. Functional restoration of VHL or molecular suppression of HIF or NF‐κB dramatically reverses CCRCC cellular susceptibility to EMCV‐induced killing. Notably, intratumoural EMCV treatment of CCRCC in a murine xenograft model rapidly regresses tumour growth. These findings provide compelling pre‐clinical evidence for the usage of EMCV in the treatment of CCRCC and potentially other tumours with elevated HIF/NF‐κB‐survival signature.

Isolation and Characterization of Cancer Stem Cells In Vitro

The cancer stem cell hypothesis is an appealing concept to account for intratumoral heterogeneity and the observation that systemic metastasis and treatment failure are often associated with the survival of a small number of cancer cells. Whilst in vivo evidence forms the foundation of this concept, in vitro methods and reagents are attractive as they offer opportunities to perform experiments that are not possible in an animal model. While there is abundant evidence that existing cancer cell lines are not reliable models of tumor heterogeneity, recent advances based on well validated novel cancer cell lines established de novo in defined serum-free media are encouraging, particularly in the study of glioblastoma multiforme. In this chapter we wish to broadly outline the process of establishing, characterizing, and managing novel cancer cell lines in defined serum-free media, and discuss the limitations and potential opportunities that may arise from these model systems.

Abstract 08: Impaired H3K36 methylation defines a subset of head and neck squamous cell carcinomas

Human papillomavirus (HPV)-negative head and neck squamous cell carcinomas (HNSCCs) are deadly and common cancers. Recent genomic studies implicate multiple genetic pathways, including cell signaling, cell cycle and immune evasion, in their development. Here we analyze public data sets and uncover a previously unappreciated role of epigenome deregulation in the genesis of 13% of HPV-negative HNSCCs. Specifically, we identify novel recurrent mutations encoding p.Lys36Met (K36M) alterations in multiple H3 histone genes. We further validate the presence of these alterations in multiple independent HNSCC data sets and show that, along with previously described NSD1 mutations, they correspond to a specific DNA methylation cluster. The K36M substitution and NSD1 defects converge on altering methylation of histone H3 at K36 (H3K36), subsequently blocking cellular differentiation and promoting oncogenesis. Our data further indicate limited redundancy for NSD family members in HPV-negative HNSCCs and suggest a potential role for impaired H3K36 methylation in their development. Further investigation of drugs targeting chromatin regulators is warranted in HPV-negative HNSCCs driven by aberrant H3K36 methylation. Citation Format: Chao Lu, Simon Papillon-Cavanagh, Tenzin Gayden, Leonie G. Mikael, Denise Bechet, Christina Karamboulas, Laurie Ailles, Jason Karamchandani, Dylan M. Marchione, Benjamin A. Garcia, Ilan Weinreb, David Goldstein, Peter W. Lewis, Octavia-Maria Dancu, Sandeep Dhaliwal, William Stecho, Christopher J. Howlett, Joe S. Mymryk, John W. Barrett, Anthony C. Nichols, C David Allis, Jacek Majewski, Nada Jabado. Impaired H3K36 methylation defines a subset of head and neck squamous cell carcinomas [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 08.

Abstract 3019: High throughput drug screening identified spleen tyrosine kinase as a novel therapeutic target in head and neck cancer with potent in vitro and in vivo activity

Background and Significance: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, and survival remains poor highlighting the need for novel treatments for the treatment of HNSCC. High throughput drug screening has shown the potential to discover novel therapeutics in other cancers. Methods: Twenty-eight HNSCC cell lines, including 5 HPV-positive lines, were characterized with whole exome sequencing and copy number arrays and screened with 1505 potential anti-cancer agents on a robotic liquid handling platform at a single dose (4uM). The most potent hits were confirmed with 10-point dose response curves. Novel therapeutic targets were further investigated with mechanistic and xenograft studies. Results: Drug screening identified 10 agents with broad activity across our cell line panel. One of the most potent agents was ER27319 maleate, reported to be a spleen tyrosine kinase (Syk) inhibitor. We confirmed that this molecule inhibited Syk phosphorylation specifically at tyrosine residues 525/526. Additionally, ER27319 maleate as well as a more clinically relevant Syk inhibitor, Fostamatinib, were observed to significantly impaired cellular migration and invasion. Additionally, siRNA knockdown of Syk in HNSCC cells was found to decrease HNSCC cell line growth. Finally, inhibition of Syk was observed to control HNSCC tumour growth in vivo in cell line-derived xenografts. Conclusions: High throughput drug screening of HNSCC cell lines identified Syk as a novel target and confirmed potent in vitro and in vivo activity. Further preclinical evaluation is planned with a panel of patient-derived xenografts. Should these results yield significant activity, we will aim to repurpose approved Syk inhibitors to improve outcomes for patients suffering from HNSCC. Citation Format: Morgan Black, Laurie Ailles, Ren Sun, Alessandro Datti, Frederick Vizeacoumar, Nicole Pinto, Kara Ruicci, John Yoo, Kevin Fung, Danielle MacNeil, David A. Palma, Eric Winquist, Joe S. Mymryk, Paul C. Boutros, John W. Barrett, Anthony C. Nichols. High throughput drug screening identified spleen tyrosine kinase as a novel therapeutic target in head and neck cancer with potent in vitro and in vivo activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3019.

Abstract 1804: The role of adipose derived stromal cells for reversal of radiation fibrosis

Hypothesis: Up to 70% of patients after radiotherapy develop radiation fibrosis (RF), an irreversible scarring of normal tissue resulting in functional morbidity and increased risk of surgical complications. Adipose-derived stromal cells (ADSCs) have been used effectively for the treatment of complex wounds in animal models and clinical trials. We hypothesize that ADSCs may reverse RF by repopulating mesenchymal precursor cells and by providing angiogenic, anti-inflammatory, and matrix remodeling factors. Methods: A RF animal model was developed and ADSC isolation was confirmed by immunophenotype and differentiation assay. GFP and luciferase labelled ADSCs were used to assess biodistribution after transplantation. To determine the therapeutic effect of ADSC transplantation for RF, we assessed functional changes to leg contracture, oxygen saturation and perfusion and molecular changes to inflammation, vascularization, and matrix remodeling. To determine the mechanism of ADSC-mediated fibrosis reversal, we assessed transcriptomic and epigenetic changes to RF tissue. Results: A RF model was created by radiating the hind limb of C3H mice. This model showed a dose dependent leg contracture and histological findings of fibrosis. We confirmed the immunophenotype of isolated ADSC and their ability to differentiate into adipogenic, chondrogenic, and osteogenic lineages. ADSC transplantation showed a statistically significant trend towards improved leg contracture (2-way ANOVA, p Conclusions: ADSC transplantation may be an effective treatment for the reversal of radiation fibrosis. As cancer survivorship increases, the prevalence of radiation fibrosis will rise and necessitate increased focus on effective treatment strategies for this condition. Citation Format: Xiao Zhao, Ju Hee Lee, Kenneth Yip, Laurie Ailles, Fei-Fei Liu. The role of adipose derived stromal cells for reversal of radiation fibrosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1804. doi:10.1158/1538-7445.AM2015-1804

Abstract 4369: The microRNA-218-survivin axis regulates cervical cancer cell migration and invasion

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA MicroRNA (miR)-218 down-regulation has been reported in numerous human malignancies. In cervical cancer, we identified that lower miR-218 expression was significantly associated with poorer overall survival, disease-free survival, and pelvic/para-aortic lymph node recurrence. Further analyses of cervical cancer data from The Cancer Genome Atlas (TCGA) identified that this down-regulation was associated with a genomic locus loss (hsa-mir-218-1:4p15.31, hsa-mir-218-2:5q34, n=105). The objective of the current study was to elucidate the cellular and molecular functions of miR-218. MiR-218 transfection into cervical cancer cells (SiHa and ME-180) significantly reduced cell migration (by 66% and 89%, respectively), invasion (by 49% and 67%, respectively), and clonogenic capacity (by 42% and 53%, respectively), relative to control-transfected cells (P<0.05). In order to identify clinically relevant miR-218 target genes, we used an integrated trimodal approach, incorporating DNA microarray (Affymetrix Human Genome U133 Plus 2.0) analyses of 79 clinical samples, miR-218 transfection, and miRDB target prediction. The most significant target was survivin (BIRC5); miR-218 transfection confirmed a reduction in survivin mRNA and protein expression in both SiHa and ME-180 cells. Furthermore, a direct interaction between the survivin-3′UTR and miR-218 was validated using a luciferase reporter assay. siRNA knockdown of survivin in SiHa and ME-180 significantly reduced cell migration (by 76% and 83%, respectively), invasion (by 79% and 88%, respectively), and clonogenic capacity (by 98% and 97%, respectively), relative to control cells (P<0.05). YM155, a small-molecule survivin suppressant, effectively reduced survivin mRNA and protein levels in a concentration- and time-dependent manner. This compound correspondingly decreased cervical cancer cell proliferation and clonogenic survival. Our results suggest that the miR-218-survivin axis plays an important role in cervical cancer progression. Citation Format: Ryunosuke Kogo, Christine How, Jeff Bruce, Willa Shi, Kenneth W. Yip, Laurie Ailles, Fei-Fei Liu. The microRNA-218-survivin axis regulates cervical cancer cell migration and invasion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4369. doi:10.1158/1538-7445.AM2014-4369

Abstract 3476: Profiling CD antigens signature in human hepatocellular carcinoma (HCC) by cell-based high-throughput screening flow cytometry (HTS-FC)

Accumulating evidence supports the cancer stem cell (CSC) hypothesis, which posits that CSCs are the source of many solid tumor types including hepatocellular carcinoma (HCC). In HCC, CSCs have been identified by a variety of CD antigens, including CD133, CD326 (EpCAM), CD90, CD44, CD24, CD13, which could serve as potential therapeutic targets. However, due to the heterogeneity of HCC, these markers do not account for CSC activity in all tumors, and there is thus a need to identify novel HCC CSC markers. We have developed a cell-based high-throughput screening flow cytometry (HTS-FC) platform to robustly characterize expression profiles of CD antigens on primary human HCC cells. We titrated all commercial fluorescence conjugated CD antibodies (CD1 to CD363) into four 96-well microplates, isolated and stained HCC cells with CD45 antibody then divided and incubated these cells with different CD antibodies in microplates (1X10^5 cells/well) followed by a LIVE/DEAD® reactive dye treatment. Stained plates were fixed and subjected to BD™ LSR II flow cytometry using High Throughput Sampler Unit. Each CD antigen expression percentage was analyzed within the CD45 negative subpopulation by FlowJo software. After investigating 10 HCC tumors, we performed heat-map and cluster analysis using Multi Experiment Viewer software. Our analysis of primary human HCC cells demonstrated that previously identified index markers of HCC CSC are expressed on a highly variable fraction of tumor cells as follows: CD13 (76.10 ± 8.72%), CD24 (7.79 ± 5.33%), CD90 (5.29 ± 0.84%), EpCAM (0.50 ± 0.46%), CD44 (0.041 ± 0.019%) and CD133 (0.0051 ± 0.0034%). Based on the heat-map, we classified all CD markers into five groups: 20 CD13-like high expressions (30%∼100%), 12 median expressions (10%∼30%), 32 CD90-like low expressions (1.0%∼10%), 60 EpCAM-like rare expressions (0.03%∼1.0%) and 249 nonsense expressions. We focused on the CD90- and EpCAM-like groups (92 CD candidates) for novel potential CSC markers. Using quantitative real-time RT-PCR, we determined the mRNA level of 92 antigens in reference to the EpCAM+/CD133+ CSC subpopulations sorted from 4 human HCC cell lines (HepG2, Hep3B, PLC/PRF/5 and Huh7) and identified 20% of these 92 candidates with stem/progenitor mRNA phenotype. We then measured the mRNA level of these remaining candidates in 40 pairs of primary human HCC tumor/adjacent normal tissues and identified two novel CD antigens showing high tumor specificity. We are now purifying these cells from primary HCC tumors in order to determine whether these small populations identify cells with CSC activity in xenograft models and sphere-forming assays. These studies demonstrate that cell-based high-throughput screening flow cytometry screening can be used in combination with other cell/molecular biological techniques to identify novel populations of tumor cells with unique functional characteristics. Citation Format: Kui Chen, Laurie Ailles, John E. Dick, Anand Ghanekar. Profiling CD antigens signature in human hepatocellular carcinoma (HCC) by cell-based high-throughput screening flow cytometry (HTS-FC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3476. doi:10.1158/1538-7445.AM2014-3476