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CRISPR activation screen in mice identifies novel membrane proteins enhancing pulmonary metastatic colonisation.

ABSTRACT: Melanoma represents ~5% of all cutaneous malignancies, yet accounts for the majority of skin cancer deaths due to its propensity to metastasise. To develop new therapies, novel target molecules must to be identified and the accessibility of cell surface proteins makes them attractive targets. Using CRISPR activation technology, we screened a library of guide RNAs targeting membrane protein-encoding genes to identify cell surface molecules whose upregulation enhances the metastatic pulmonary colonisation capabilities of tumour cells in vivo. We show that upregulated expression of the cell surface protein LRRN4CL led to increased pulmonary metastases in mice. Critically, LRRN4CL expression was elevated in melanoma patient samples, with high expression levels correlating with decreased survival. Collectively, our findings uncover an unappreciated role for LRRN4CL in the outcome of melanoma patients and identifies a potential therapeutic target and biomarker.

SUBMITTER: van der Weyden L

PROVIDER: S-EPMC7987976 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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CRISPR activation screen in mice identifies novel membrane proteins enhancing pulmonary metastatic colonisation.

van der Weyden Louise L Harle Victoria V Turner Gemma G Offord Victoria V Iyer Vivek V Droop Alastair A Swiatkowska Agnieszka A Rabbie Roy R Campbell Andrew D AD Sansom Owen J OJ Pardo Mercedes M Choudhary Jyoti S JS Ferreira Ingrid I Tullett Mark M Arends Mark J MJ Speak Anneliese O AO Adams David J DJ

Communications biology 20210323 1

Melanoma represents ~5% of all cutaneous malignancies, yet accounts for the majority of skin cancer deaths due to its propensity to metastasise. To develop new therapies, novel target molecules must to be identified and the accessibility of cell surface proteins makes them attractive targets. Using CRISPR activation technology, we screened a library of guide RNAs targeting membrane protein-encoding genes to identify cell surface molecules whose upregulation enhances the metastatic pulmonary colo ...[more]

PMID: 33758365

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Project description:Zika virus (ZIKV) is an arthropod-borne emerging pathogen causing febrile illness. ZIKV is associated Guillain-Barré syndrome and other neurological complications. Infection during pregnancy is associated with pregnancy complications and developmental and neurological abnormalities collectively defined as congenital Zika syndrome. There is still no vaccine or specific treatment for ZIKV infection. To identify host factors that can rescue cells from ZIKV infection, we used a genome-scale CRISPR activation screen. Our highly ranking hits included a short list of interferon-stimulated genes (ISGs) previously reported to have antiviral activity. Validation of the screen results highlighted interferon lambda 2 (IFN-λ2) and interferon alpha-inducible protein 6 (IFI6) as genes providing high levels of protection from ZIKV. Activation of these genes had an effect on an early stage in viral infection. In addition, infected cells expressing single guide RNAs (sgRNAs) for both of these genes displayed lower levels of cell death than did the controls. Furthermore, the identified genes were significantly induced in ZIKV-infected placenta explants. Thus, these results highlight a set of ISGs directly relevant for rescuing cells from ZIKV infection or its associated cell death and substantiate CRISPR activation screens as a tool to identify host factors impeding pathogen infection.IMPORTANCE Zika virus (ZIKV) is an emerging vector-borne pathogen causing a febrile disease. ZIKV infection might also trigger Guillain-Barré syndrome, neuropathy, and myelitis. Vertical transmission of ZIKV can cause fetus demise, stillbirth, or severe congenital abnormalities and neurological complications. There is no vaccine or specific antiviral treatment against ZIKV. We used a genome-wide CRISPR activation screen, where genes are activated from their native promoters to identify host cell factors that protect cells from ZIKV infection or associated cell death. The results provide a better understanding of key host factors that protect cells from ZIKV infection and might assist in identifying novel antiviral targets.

Project description:Metastasis is the leading cause of death in cancer patients, and successful colonisation of a secondary organ by circulating tumour cells (CTCs) is the rate-limiting step of this process. We used tail-vein injection of B16-F10 melanoma cells into mice to mimic the presence of CTCs and to allow for the assessment of host (microenvironmental) factors that regulate pulmonary metastatic colonisation. We found that mice deficient for the individual subunits of the NADPH oxidase of myeloid cells, NOX2 (encoded by Cyba, Cybb, Ncf1, Ncf2, and Ncf4), all showed decreased pulmonary metastatic colonisation. To understand the role of NOX2 in controlling tumour cell survival in the pulmonary microenvironment, we focused on Cyba-deficient (Cybatm1a ) mice, which showed the most significant decrease in metastatic colonisation. Interestingly, histological assessment of pulmonary metastatic colonisation was not possible in Cybatm1a mice, owing to the presence of large granulomas composed of galectin-3 (Mac-2)-positive macrophages and eosinophilic deposits; granulomas of variable penetrance and severity were also found in Cybatm1a mice that were not injected with melanoma cells, and these contributed to their decreased survival. The decreased pulmonary metastatic colonisation of Cybatm1a mice was not due to any overt defects in vascular permeability, and bone marrow chimaeras confirmed a role for the haematological system in the reduced metastatic colonisation phenotype. Examination of the lymphocyte populations, which are known key regulators of metastatic colonisation, revealed an enhanced proportion of activated T and natural killer cells in the lungs of Cybatm1a mice, relative to controls. The reduced metastatic colonisation, presence of granulomas and altered immune cell populations observed in Cybatm1a lungs were mirrored in Ncf2-deficient (Ncf2tm1a ) mice. Thus, we show that NOX2 deficiency results in both granulomas and the accumulation of antitumoural immune cells in the lungs that probably mediate the decreased pulmonary metastatic colonisation. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Dataset Information

CRISPR activation screen in mice identifies novel membrane proteins enhancing pulmonary metastatic colonisation.

Publications

CRISPR activation screen in mice identifies novel membrane proteins enhancing pulmonary metastatic colonisation.

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