Identification of Novel Regulators of the Hippo Pathway using Positive Selection Genome Wide CRISPR Screens
Bibliographic record
Abstract
The Hippo pathway is established as a master regulator of tissue growth and organ size and its dysregulation can lead to cancer. The core pathway consists of the kinases MST1/2 and LATS1/2, their adaptor proteins, SAV1 and MOB1A/1B, respectively, and the transcriptional module YAP and TAZ. Multiple upstream signals such as mechanotransduction, GPCRs and metabolic pathways activate MST1/2 which along with SAV phosphorylates and activates LATS1/2 together with MOB proteins which phosphorylate and sequester YAP/TAZ in the cytoplasm leading to their subsequent degradation. The core pathway is well studied, however, many upstream pathway regulators are ill-defined/unknown. To identify novel Hippo pathway regulators, I undertook two positive selection genome-wide CRISPR screens. I generated clonal cell lines of T24 bladder and MDA-MB-231 breast cancer cells expressing two proteins, Cas9 which enabled gene deletion and a FKBPiCaspase9 fusion protein under YAP/TAZ transcriptional control, which when expressed causes cell death upon addition of chemical dimerizer, AP20187. CRISPR-mediated loss of genes promoting YAP/TAZ activity, allows cell survival by downregulating the FKBPiCaspase9 protein. The first screen, in T24 bladder cancer cells identified the phosphatase adaptor protein PPFIA1 as a positive regulator of YAP/TAZ. I demonstrated that loss of PPFIA1 leads to decreased YAP/TAZ protein levels, increased YAP/TAZ phosphorylation, enhanced cytoplasmic localization and iii decreased YAP/TAZ target gene expression. Immunoprecipitation analysis demonstrated that PPFIA1 interacts with YAP. These data suggest a model in which PPFIA1 plays a role in maintaining YAP in a dephosphorylated state, possibly by recruiting a phosphatase. To identify novel regulators that might be relevant to multiple cell lines I performed a second screen in the MDA-MB-231 cells and identified the Gα protein chaperone RIC8A, which also functions as a Guanine Nucleotide Exchange Factor (GEF), as another putative regulator of the Hippo pathway. I showed that abrogation of RIC8A expression mediated a decrease in total YAP /TAZ protein levels, a decrease in YAP /TAZ target gene expression and increased cytoplasmic localization of YAP/TAZ in both bladder and breast cancer cells. Overall, my thesis introduces a novel method to interrogate the Hippo pathway in multiple cancer types and identifies new regulators of YAP and TAZ.
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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) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.001 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.003 | 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".