A Genome‐Wide CRISPR‐Cas9 Screen Identifies Importin‐β11 as a Required Factor for β‐catenin Signaling in Colon Cancer
Bibliographic record
Abstract
Over 25000 Canadians were diagnosed with colorectal cancer last year. The majority of colorectal adenocarcinomas (CRC) have a mutation in APC (adenomatous polyposis coli), a key component of the Wnt signaling pathway. In the absence of Wnt, APC is part of a protein complex that marks the Wnt signaling effector, β‐catenin, for ubiquitin‐mediated proteolysis. In the presence of Wnt ligands, β‐catenin escapes degradation, accumulates in the nucleus and regulates context‐dependent transcriptional programs regulating stem cell renewal, proliferation and differentiation. Inactivating APC mutations lead to hyperactive Wnt‐ β‐catenin signaling, driving tumor initiation and growth. Our objective was to identify novel regulators of the β‐catenin signaling pathway downstream of APC mutation in the context of colon cancer. We have developed a positive‐selection genetic screen to identify genes required for Wnt‐ β‐catenin signal transduction. This system leverages the DLD‐1 colon cancer cell line, which contains an inactivating APC mutation and therefore has high constitutive Wnt signaling, and an inducible synthetic reporter gene system that leads to apoptotic cell death when the Wnt‐ β‐catenin signaling pathway is activated. This reporter consists of β‐catenin‐ responsive LEF‐TCF elements driving the expression of an inducible version of Caspase9, a member of the intrinsic apoptotic cascade. To identify genes required for β‐catenin signaling in these cells, a CRISPR‐Cas9 functional screen was performed. This genome‐wide screen applies the TKO sgRNA lentiviral library that contains over 90000 unique sgRNAs that target over 17500 human genes. Upon induction of Caspase9 activity, only cells harbouring loss‐of‐function within genes essential for Wnt‐ β‐catenin signaling are able to grow. The sgRNA conferring growth‐rescue were amplified from genomic DNA and identified using next generation sequencing. The top genes enriched in the resistant cell population included known members of the Wnt‐ β‐catenin signaling pathway as well as a gene, which has no previous link to the Wnt signaling pathway, IPO11 . Knockout of IPO11 confirmed its requirement for Wnt‐ β‐catenin signal transduction by luciferase assay and qPCR of β‐catenin target genes. IPO11 encodes for a nuclear import protein, importin‐ β11, which transports cargo into the nucleus. Knockout of IPO11 reduces β‐catenin nuclear localization. Importin‐ β11 associates with β‐catenin in cells, and this interaction is competed by dominant‐negative Ran mutant (Q69L). Knock‐out of IPO11 reduces proliferation of patient‐ derived, colorectal cancer cells, three‐dimensional organoid model, which mimics the physiological conditions required for cancer stem cell maintenance. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".