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Image-based profiling has emerged as a powerful technology for various steps in basic biological and pharmaceutical discovery, but the community has lacked a large, public reference set of data from chemical and genetic perturbations. Here we present data generated by the Joint Undertaking for Morphological Profiling (JUMP)-Cell Painting Consortium, a collaboration between 10 pharmaceutical companies, six supporting technology companies, and two non-profit partners. When completed, the dataset will contain images and profiles from the Cell Painting assay for over 116,750 unique compounds, over-expression of 12,602 genes, and knockout of 7,975 genes using CRISPR-Cas9, all in human osteosarcoma cells (U2OS). The dataset is estimated to be 115 TB in size and capturing 1.6 billion cells and their single-cell profiles. File quality control and upload is underway and will be completed over the coming months at the Cell Painting Gallery:https://registry.opendata.aws/cellpainting-gallery. A portal to visualize a subset of the data is available athttps://phenaid.ardigen.com/jumpcpexplorer/.

Cell plasticity is a crucial hallmark leading to cancer metastasis. Upregulation of Rho/ROCK pathway drives actomyosin contractility, protrusive forces, and contributes to the occurrence of highly invasive amoeboid cells in tumors. Cancer stem cells are similarly associated with metastasis, but how these populations arise in tumors is not fully understood. Here, we show that the novel oncogene RASSF1C drives mesenchymal‐to‐amoeboid transition and stem cell attributes in breast cancer cells. Mechanistically, RASSF1C activates Rho/ROCK via SRC‐mediated RhoGDI inhibition, resulting in generation of actomyosin contractility. Moreover, we demonstrate that RASSF1C‐induced amoeboid cells display increased expression of cancer stem‐like markers such as CD133, ALDH1, and Nanog, and are accompanied by higher invasive potential in vitro and in vivo. Further, RASSF1C‐induced amoeboid cells employ extracellular vesicles to transfer the invasive phenotype to target cells and tissue. Importantly, the underlying RASSF1C‐driven biological processes concur to explain clinical data: namely, methylation of the RASSF1C promoter correlates with better survival in early‐stage breast cancer patients. Therefore, we propose the use of RASSF1 gene promoter methylation status as a biomarker for patient stratification., Tumour‐suppressor RASSF1A encodes an alternative transcript, RASSF1C, that acts as oncogene in human breast cancer, triggering pro‐invasive cellular features and aggressiveness.

Cell plasticity is a crucial hallmark leading to cancer metastasis. Upregulation of Rho/ROCK pathway drives actomyosin contractility, protrusive forces and contributes to the occurrence of highly invasive amoeboid cells in tumors. Cancer stem cells are similarly associated with metastasis, but how these populations arise in tumors is not fully understood. Here we show that the novel oncogene RASSF1C drives mesenchymal to amoeboid transition and stem cell attributes in breast cancer cells. Mechanistically, RASSF1C activates Rho/ROCK via SRC mediated RhoGDI inhibition, resulting in generation of actomyosin contractility. Moreover, we demonstrate that amoeboid cells display the cancer stem cell markers CD133, ALDH1 and the pluripotent marker Nanog; are accompanied by higher invasive potential in vitro and in vivo; and employ extracellular vesicles to transfer the invasive phenotype to target cells and tissue. Importantly, the underlying RASSF1C driven biological processes concur to explain clinical data: namely, methylation of the RASSF1C promoter correlates with better survival in early stage breast cancer patients. Therefore, we propose the use of RASSF1 gene promoter methylation status as a biomarker for patient stratification.

Epigenetic inactivation of the Hippo pathway scaffold RASSF1A is associated with poor prognosis in a wide range of sporadic human cancers. Loss of expression reduces tumor suppressor activity and promotes genomic instability, but how this pleiotropic biomarker is regulated at the protein level is unknown. Here we show that TGF-β is the physiological signal that stimulates RASSF1A degradation by the ubiquitin-proteasome pathway. In response to TGF-β, RASSF1A is recruited to TGF-β receptor I and targeted for degradation by the co-recruited E3 ubiquitin ligase ITCH. RASSF1A degradation is necessary to permit Hippo pathway effector YAP1 association with SMADs and subsequent nuclear translocation of receptor-activated SMAD2. We find that RASSF1A expression regulates TGF-β-induced YAP1/SMAD2 interaction and leads to SMAD2 cytoplasmic retention and inefficient transcription of TGF-β targets genes. Moreover, RASSF1A limits TGF-β induced invasion, offering a new framework on how RASSF1A affects YAP1 transcriptional output and elicits its tumor-suppressive function.

Summary Tumor progression to invasive carcinoma is associated with activation of SRC family kinase (SRC, YES, FYN) activity and loss of cellular cohesion. The hippo pathway-regulated cofactor YAP1 supports the tumorigenicity of RAS mutations but requires both inactivation of hippo signaling and YES-mediated phosphorylation of YAP1 for oncogenic activity. Exactly how SRC kinases are activated and hippo signaling is lost in sporadic human malignancies remains unknown. Here, we provide evidence that hippo-mediated inhibition of YAP1 is lost upon promoter methylation of the RAS effector and hippo kinase scaffold RASSF1A. We find that RASSF1A promoter methylation reduces YAP phospho-S127, which derepresses YAP1, and actively supports YAP1 activation by switching RASSF1 transcription to the independently transcribed RASSF1C isoform that promotes Tyr kinase activity. Using affinity proteomics, proximity ligation, and real-time molecular visualization, we find that RASSF1C targets SRC/YES to epithelial cell-cell junctions and promotes tyrosine phosphorylation of E-cadherin, β-catenin, and YAP1. RASSF1A restricts SRC activity, preventing motility, invasion, and tumorigenesis in vitro and in vivo, with epigenetic inactivation correlating with increased inhibitory pY527-SRC in breast tumors. These data imply that distinct RASSF1 isoforms have opposing functions, which provide a biomarker for YAP1 activation and explain correlations of RASSF1 methylation with advanced invasive disease in humans. The ablation of epithelial integrity together with subsequent YAP1 nuclear localization allows transcriptional activation of β-catenin/TBX-YAP/TEAD target genes, including Myc, and an invasive phenotype. These findings define gene transcript switching as a tumor suppressor mechanism under epigenetic control., Graphical Abstract, Highlights • Methylation of RASSF1A correlates with loss of hippo-inhibitory phospho-S127-YAP1 • SRC, FYN, and YES are activated by RASSF1C in RASSF1A-methylated cells • RASSF1C promotes E-cadherin internalization and reduces cell junction integrity • RASSF1A loss drives RASSF1C-YAP1/β-catenin-mediated transcription and invasion, In a wide variety of sporadic malignancies, promoter methylation of the RASSF1 gene is associated with tumor invasion and metastasis. Vlahov et al. show that the clinical phenotype is driven by both RASSF1A loss and the independently transcribed RASSF1C isoform, which promotes SRC activation, pseudo-EMT, and β-catenin/YAP1-dependent invasion of tumor cells.

Investigation of the mechanisms behind the regulation of cellular motility and adhesion is key to understanding metastasis and the biology of tumor spreading. There are many technologies available for these studies, but the majority of them are either dependent on the use of labels or limited to endpoint analysis. The xCELLigence RTCA (real-time cell analysis) provides a platform for label free and operator independent investigation of the migration, invasion and adhesion proprieties of cells in physiologically relevant conditions. The real-time kinetic data acquisition also allows for a more accurate characterization of short-lived cellular events. In this chapter we describe the use of the xCELLigence Real-Time Cell Analyzer to investigate changes in cellular adhesion and motility in real time. © Springer Science+Business Media, LLC 2013.

SummaryHypermethylation of CpG islands in the RASSF1 promoter is one of the most frequent events identified in human cancer [1–3]. The epigenetic-driven loss of RASSF1A protein expression is observed more often in tumors of higher grade and correlates with a decreased responsiveness to DNA-damaging therapy [4–6]. Ras association domain-containing family 1A (RASSF1A) promotes apoptosis by signaling through the MST2 and LATS1 kinases, leading to stabilization of the YAP1/p73 transcriptional complex [7]. Here we provide evidence for a new pathway linking DNA damage signaling to RASSF1A via the main sensor of double-strand breaks in cells, ataxia telangiectasia mutated (ATM). We show that, upon DNA damage, RASSF1A is phosphorylated by ATM on Ser131 and is involved in the activation of both MST2 and LATS1, leading to the stabilization of p73. Furthermore, lung and ovarian tumor cell lines that retain RASSF1A expression commonly harbor polymorphisms in the region of Ser131 [8], and our analysis shows that the S131F polymorphism conveys resistance to DNA-damaging agents. Thus, we present a novel DNA damage pathway emanating from ATM that is frequently disabled in tumors via epigenetic silencing of RASSF1 or mutation of an ATM phosphorylation site.

// Manuel Sarmiento Soto 1 , Emma R. O’Brien 1 , Kleopatra Andreou 1 , Simon F. Scrace 1 , Rasheed Zakaria 2 , Michael D. Jenkinson 2, 3 , Eric O’Neill 1 , Nicola R. Sibson 1 1 Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford OX3 7LE, UK 2 Institute of Integrative Biology, University of Liverpool, Liverpool, L69 3BX, UK 3 Department of Neurosurgery, The Walton Centre NHS Foundation Trust, Liverpool, L97LJ, UK Correspondence to: Nicola R. Sibson, email: nicola.sibson@oncology.ox.ac.uk Keywords: brain metastasis, cellular adhesion molecules, LFA-1, COX-2, eNOS Received: October 07, 2015 Accepted: June 26, 2016 Published: July 20, 2016 ABSTRACT Over 20% of cancer patients will suffer metastatic spread to the brain, and prognosis remains poor. Communication between tumour cells and host tissue is essential during metastasis, yet little is known of the processes underlying such interactions in the brain. Here we test the hypothesis that cross-talk between tumour cells and host brain cells, through tumour cell leukocyte function associated protein-1 (LFA-1), is critical in metastasis development. Temporal expression of LFA-1 and its major ligand intercellular adhesion molecule-1 (ICAM-1) was determined in two different mouse models of brain metastasis. Marked upregulation of both proteins was found, co-localising with astrocytes, microglia and tumour cells themselves. Silencing of LFA-1 expression in MDA231Br-GFP cells prior to intracerebral injection resulted in > 70% reduction in tumour burden compared to control MDA231Br-GFP cells ( p < 0.005, n = 5). Subsequent qRT-PCR analysis of brain tissue revealed significant reductions in COX-2, VEGF and eNOS from host brain tissue, but not tumour cells, in mice injected with LFA-1 knockdown cells ( p < 0.0001, n = 5). Finally, expression of both LFA-1 and ICAM-1 was demonstrated in human brain metastasis samples. The results of this study suggest LFA-1 as a new target in brain metastasis therapy and highlight the potential synergy with current anti-COX-2 and anti-NOS therapies.

// Andrew J. Massey 1 , Stephen Stokes 1 , Helen Browne 1 , Nicolas Foloppe 1 , Andrea Fiumana 1 , Simon Scrace 1,3 , Mandy Fallowfield 1 , Simon Bedford 1 , Paul Webb 1 , Lisa Baker 1 , Mark Christie 2 , Martin J. Drysdale 1,4 and Mike Wood 1 1 Vernalis Research, Granta Park, Cambridge, UK 2 Akranim Ltd, Maidstone, UK 3 Horizon Discovery, Cambridge Research Park, Waterbeach, Cambridge, UK 4 Cancer Research UK Beatson Institute, Garscube Estate, Bearsden, Glasgow, UK Correspondence to: Andrew J. Massey, email: // Keywords : Chk1, V158411, fragment, drug discovery, colon cancer Received : June 17, 2015 Accepted : September 14, 2015 Published : September 30, 2015 Abstract Chk1 kinase is a critical component of the DNA damage response checkpoint especially in cancer cells and targeting Chk1 is a potential therapeutic opportunity for potentiating the anti-tumor activity of DNA damaging chemotherapy drugs. Fragment elaboration by structure guided design was utilized to identify and develop a novel series of Chk1 inhibitors culminating in the identification of V158411, a potent ATP-competitive inhibitor of the Chk1 and Chk2 kinases. V158411 abrogated gemcitabine and camptothecin induced cell cycle checkpoints, resulting in the expected modulation of cell cycle proteins and increased cell death in cancer cells. V158411 potentiated the cytotoxicity of gemcitabine, cisplatin, SN38 and camptothecin in a variety of p53 deficient human tumor cell lines in vitro , p53 proficient cells were unaffected. In nude mice, V158411 showed minimal toxicity as a single agent and in combination with irinotecan. In tumor bearing animals, V158411 was detected at high levels in the tumor with a long elimination half-life; no pharmacologically significant in vivo drug-drug interactions with irinotecan were identified through analysis of the pharmacokinetic profiles. V158411 potentiated the anti-tumor activity of irinotecan in a variety of human colon tumor xenograft models without additional systemic toxicity. These results demonstrate the opportunity for combining V158411 with standard of care chemotherapeutic agents to potentiate the therapeutic efficacy of these agents without increasing their toxicity to normal cells. Thus, V158411 would warrant further clinical evaluation.

REDX05358 has been identified as a novel, highly selective and potent next generation pan RAF inhibitor with improved therapeutic potential and predicted safety profile. Aberrant signalling via the MAPK pathway is commonly seen in cancer. RAF inhibitors such as Dabrafenib and Vemurafenib have been approved for the treatment of unresectable and metastatic BRAF mutant melanoma, but these agents lack efficacy in BRAF mutant colorectal cancer (CRC), partly because of EGFR-mediated feedback reactivation of the MAPK pathway via CRAF. Furthermore, RAF inhibitor treatment of RAS-mutant, BRAF wildtype melanomas has been associated with other skin cancers, such as cutaneous squamous cell carcinoma due to MAPK pathway paradoxical activation mediated by CRAF. There is therefore a clinical need for novel agents targeting the MAPK pathway that do not have these undesirable properties. Here we present, REDX05358 that demonstrates subnanomolar binding affinity for BRAF and CRAF with high selectivity profile against a panel of 468 kinases that exhibits negligible paradoxical activation due to inhibition of both RAF monomers and dimers. As a result, REDX05358 not only inhibits MAPK signalling in BRAF V600E mutant tumor cells, but also in those harbouring NRAS and KRAS mutations. Furthermore, REDX05358 does not induce feedback reactivation of the pathway through its ability to sustain inhibition of MAPK signalling in CRC cell lines. Correspondingly, profiling of REDX05358 in a panel of CRC, melanoma and NSCLC cell lines shows it has potent anti-proliferative activity in cell lines harbouring BRAF or RAS mutations. REDX05358 is an orally bioavailable, well tolerated small molecule that has demonstrated in vivo efficacy in BRAF V600E CRC xenograft model. In contrast, first generation inhibitors such as Vemarafenib and Dabrafanib have been reported to be ineffective in this genetic background in CRC cell lines and patients. Thus, we have developed a pan RAF inhibitor with unique pharmacological properties enabling it to have utility in treating BRAF and RAS mutant cancers Citation Format: Helen Mason, Simon Scrace, Richard Testar, Julie Rainard, Fatima Talab, Ryaka Poonawala, Philippa Smith, Harriet Brooke, Sarah Frith, Jonathan Ahmet, Jonathan Hall, David Sorrell, Jon Moore, Caroline Phillips, Dennis France, Matilda Bingham, Richard Armer. Development of REDX05358, a novel highly selective and potent pan RAF inhibitor and a potential therapeutic for BRAF and RAS mutant tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5160. doi:10.1158/1538-7445.AM2017-5160

Investigation of the mechanisms behind the regulation of cellular motility and adhesion is key to understanding metastasis and the biology of tumor spreading. There are many technologies available for these studies, but the majority of them are either dependent on the use of labels or limited to endpoint analysis. The xCELLigence RTCA (real-time cell analysis) provides a platform for label free and operator independent investigation of the migration, invasion and adhesion proprieties of cells in physiologically relevant conditions. The real-time kinetic data acquisition also allows for a more accurate characterization of short-lived cellular events. In this chapter we describe the use of the xCELLigence Real-Time Cell Analyzer to investigate changes in cellular adhesion and motility in real time.

The RASSF family of proteins has been extensively studied in terms of their genetics, structure and function. One of the functions that has been increasingly studied is the role of the RASSF proteins in the DNA damage response. Surprisingly, this research, which encompasses both the classical and N-terminal RASSF proteins, has revealed an involvement of the RASSFs in oncogenic pathways as well as the more familiar tumour suppressor pathways usually associated with the RASSF family members. The most studied protein with respect to DNA damage is RASSF1A, which has been shown, not only to be activated by ATM, a major regulator of the DNA damage response, but also to bind to and activate a number of different pathways which all lead to and feedback from the guardian of the genome, p53. In this review we discuss the latest research linking the RASSF proteins to DNA damage signalling and maintenance of genomic integrity and look at how this knowledge is being utilised in the clinic to enhance the effectiveness of traditional cancer therapies such as radiotherapy.