CRISPR Activation Screening of Circulating Tumor Cells Identifies Enhancers of Blood-Based Metastasis

breast cancer;metastasis;circulating tumor cells;CRISPR activation;ribosome;circadian clock

Ebright, Richard Yon ; Mostoslavsky, Raul

University:Havard University

Department:Medical Sciences

关键词: breast cancer;    metastasis;    circulating tumor cells;    CRISPR activation;    ribosome;    circadian clock;   

Others  :  https://dash.harvard.edu/bitstream/handle/1/37365157/EBRIGHT-DISSERTATION-2020.pdf?sequence=1&isAllowed=y

美国|英语

来源: Digital Access to Scholarship at Harvard

【 摘 要 】

Over ninety percent of cancer mortality is attributable to metastasis, most commonly due to the blood-borne dissemination of cancer cells from a primary tumor to secondary tissues. However, the vast majority of these cancer cells in the circulation, known as circulating tumor cells (CTCs), never go on to form clinically relevant metastases, instead dying or senescing in the circulation or at distant sites. As such, identification of factors that promote the metastatic ability of CTCs will shed light upon CTC biology, as well as suggest novel targets for therapeutic intervention to prevent or slow metastatic growth. We conducted in vivo genome-wide CRISPR activation screens to identify genes capable of accelerating distal metastasis by breast cancer patient-derived circulating tumor cells following direct intravascular inoculation in mice. From these screens, we identified and conducted follow-up studies on two classes of hits: ribosomal proteins, primarily RPL15, and transcriptional regulators, primarily ID3. Expression of RPL15, a component of the large ribosome subunit, is sufficient to increase metastatic growth in multiple organs. RPL15 overexpression selectively increases translation of cell cycle regulators and other ribosomal proteins. Unsupervised analysis of single-cell RNA sequencing of freshly isolated CTCs from breast cancer patients identifies a subset with strong ribosomal and protein translation signatures, correlated with increased proliferative markers, CTC clusters and poor clinical outcome. Thus, ribosome protein expression identifies an aggressive subset of CTCs, whose therapeutic targeting may suppress metastatic progression. Expression of ID3, an inhibitor of bHLH transcription factors, is also sufficient to increase metastatic growth in multiple organs. siRNA screening of bHLH transcription factors inhibited by ID3 identifies knockdown of ARNTL2, a transcription factor involved in circadian clock signaling, as able to increase CTC growth. Clinically, ID3 and ARNTL2 expression are correlated with worse and better outcome, respectively, in triple negative breast cancer, matching our in vitro and in vivo results. Thus, ID3 and ARNTL2 represent novel genes involved in breast cancer metastasis, and dysregulation of circadian clock signaling is implicated in progression through the metastatic cascade.