Rab11-FIP1/RCP Functions as a Major Signalling Hub in the Oncogenic Roles of Mutant p53 in Cancer

Rab11-FIP1 is a Rab effector protein that is involved in endosomal recycling and trafficking of various molecules throughout the endocytic compartments of the cell. The consequence of this can be increased secretion or increased membrane expression of those molecules. In general, expression of Rab11-FIP1 coincides with more tumourigenic and metastatic cell behaviour. Rab11-FIP1 can work in concert with oncogenes such as mutant p53, but has also been speculated to be an oncogene in its own right. In this perspective, we will discuss and speculate upon our observations that mutant p53 promotes Rab11-FIP1 function to not only promote invasive behaviour, but also chemoresistance by regulating a multitude of different proteins.

Zebrafish xenografts to isolate unique human breast cancer metastatic cell populations

Cancer metastasis is a critical culprit frequently blamed for treatment failure, drug resistance, poor prognosis, and high mortality rate among all human cancers. Laboratory efforts to isolate metastatic cell populations have typically been confined to mouse models, which are time-consuming and expensive. Here, we present a model system based on xenografting zebrafish embryos to select for cells that are predisposed to progress through the early stages of metastasis. This model requires only 3-5 days to achieve distinct intravasation to the zebrafish circulatory system. The metastatic cells are easily tracked in real-time as they migrate, as well as isolated and propagated in vitro. Once expanded, molecular characterization of the serially derived invasive cell populations from the tails of the zebrafish accurately predicts genes, signaling pathways, protein-protein interactions, and differential splicing products that are important for an invasive phenotype. This zebrafish model therefore offers a high-throughput and robust method for identifying gene targets critical for cancer metastasis.

Metastatic Esophageal Carcinoma Cells Exhibit Reduced Adhesion Strength and Enhanced Thermogenesis

Despite continuous improvements in multimodal therapeutic strategies, esophageal carcinoma maintains a high mortality rate. Metastases are a major life-limiting component; however, very little is known about why some tumors have high metastatic potential and others not. In this study, we investigated thermogenic activity and adhesion strength of primary tumor cells and corresponding metastatic cell lines derived from two patients with metastatic adenocarcinoma of the esophagus. We hypothesized that the increased metastatic potential of the metastatic cell lines correlates with higher thermogenic activity and decreased adhesion strength. Our data show that patient-derived metastatic esophageal tumor cells have a higher thermogenic profile as well as a decreased adhesion strength compared to their corresponding primary tumor cells. Using two paired esophageal carcinoma cell lines of primary tumor and lymph nodes makes the data unique. Both higher specific thermogenesis profile and decreased adhesion strength are associated with a higher metastatic potential. They are in congruence with the clinical patient presentation. Understanding these functional, biophysical properties of patient derived esophageal carcinoma cell lines will enable us to gain further insight into the mechanisms of metastatic potential of primary tumors and metastases. Microcalorimetric evaluation will furthermore allow for rapid assessment of new treatment options for primary tumor and metastases aimed at decreasing the metastatic potential.

Cysteine and Folate Metabolism Are Targetable Vulnerabilities of Metastatic Colorectal Cancer

With most cancer-related deaths resulting from metastasis, the development of new therapeutic approaches against metastatic colorectal cancer (mCRC) is essential to increasing patient survival. The metabolic adaptations that support mCRC remain undefined and their elucidation is crucial to identify potential therapeutic targets. Here, we employed a strategy for the rational identification of targetable metabolic vulnerabilities. This strategy involved first a thorough metabolic characterisation of same-patient-derived cell lines from primary colon adenocarcinoma (SW480), its lymph node metastasis (SW620) and a liver metastatic derivative (SW620-LiM2), and second, using a novel multi-omics integration workflow, identification of metabolic vulnerabilities specific to the metastatic cell lines. We discovered that the metastatic cell lines are selectively vulnerable to the inhibition of cystine import and folate metabolism, two key pathways in redox homeostasis. Specifically, we identified the system xCT and MTHFD1 genes as potential therapeutic targets, both individually and combined, for combating mCRC.

MBRS-45. TWIST1 AND ABCB1 ARE FUNCTIONAL DETERMINANTS OF METASTASIS IN MEDULLOBLASTOMA

Paediatric medulloblastomas (MB) are frequently metastatic, resulting in a poor prognosis for the patient. Of the four MB subgroups, group 3 patients present with the highest rates of metastasis and worst outcomes. The mechanisms behind the metastatic process are poorly understood, limiting our ability to develop novel therapeutic treatments. We hypothesised that the epithelial-mesenchymal transition (EMT) transcription factor TWIST1 and the multidrug efflux pump ABCB1 (ATP-binding cassette subfamily B member 1) synergistically drive MB metastasis. TWIST1 protein expression was analysed in patient tissue microarrays by immunohistochemistry. High TWIST1 expression was associated with metastatic patients (p=0.041). Physical and functional interactions between TWIST1 and ABCB1 were investigated using chromatin immunoprecipitation (ChIP) and a 3D migration and invasion model. ChIP analysis confirmed TWIST1 binding to the ABCB1 promoter in SHH (ONS-76) and group 3 (D283MED and HD-MB03) metastatic cell lines. TWIST1 and ABCB1 were inhibited in HDMB03 cells with harmine and vardenafil respectively, resulting in attenuated cell migration in the 3D model. Western blot and qRT-PCR analysis of harmine treated cells confirmed a reduction in ABCB1 protein and gene expression. Overall our data reveals TWIST1 and ABCB1 to be key targets for MB metastatic disease. Using bioinformatics analysis and ChIP sequencing, additional TWIST1 downstream targets are now being identified and compared across the metastatic cell lines (ONS-76, D283MED and HD-MB03). This data will provide a deeper insight into the pathways associated with MB metastases, enabling personalised treatment approaches for patients with metastatic disease.

MBRS-28. EXOSOMES DRIVE MEDULLOBLASTOMA METASTASIS IN A MMP2 AND EMMPRIN DEPENDENT MANNER

INTRODUCTION Recurrent/metastatic medulloblastoma (MB) is a devastating disease with an abysmal prognosis of less than 10% 5-year survival. The secretion of extracellular vesicles (EVs) has emerged as a pivotal mediator for communication in the tumour microenvironment during metastasis. The most investigated EV’s are exosomes, nanovesicles secreted by all cell types and able to cross the blood-brain-barrier. Matrix metalloproteinases (MMPs) are enzymes secreted by tumour cells that can potentiate their dissemination by modification of the extracellular matrix. We hypothesise that exosomal MMP2 and its inducer EMMPRIN could enhance metastasis of MB. METHODS Proliferation, invasion and migration assays were used to evaluate the phenotypic behaviour of primary cell lines pre-treated with metastatic tumour cell-derived exosomes. Gelatin zymography and western blotting were performed to confirm MMP2 functional activity in cell lines and exosomes. Nanoscale flow cytometry was used to measure surface exosomal EMMPRIN levels. Exosomal MMP2 and EMMPRIN were modulated at the RNA level. RESULTS Number of exosomes is directly related to the migratory behaviour of parental MB cell lines (p<0.01). Notably, functional exosomal MMP2 and EMMPRIN levels also correlate with this. Furthermore, exosomes from metastatic cell lines conferred enhanced migration and invasion on their matched isogenic primary (non-metastatic) cell line pair by ~3.8-fold (p<0.01). Exosomes from metastatic cell lines also conferred increased migration on poorly migratory foetal neuronal stem cells. CONCLUSION Together this data suggests that exosomal MMP2 and EMMPRIN may promote medulloblastoma metastasis and supports analysis of exosomal MMP2 and EMMPRIN levels in patient cerebral spinal fluid samples.