Periodic Reporting for period 3 - TRANSREG (Dissecting the role of Translational Regulation in Tumorigenesis)
Periodo di rendicontazione: 2020-11-01 al 2022-04-30
The control of translation is a key determinant of protein abundance, which in turn defines cellular states. The impact of translational regulation may be even greater during the transition from homeostasis to malignancy, as revealed by the surprisingly low correlations between mRNA and protein levels in human cancer databases. This raises the intriguing possibility that through an ability to generate aberrant downstream networks of translational regulators, oncogenic drivers might impose altered protein synthesis programs that become the driving force for tumor formation and malignant progression.
We recently unveiled a hitherto unappreciated role for upstream open reading frame (uORF) translation in tumorigenesis and unearthed a novel switch from conventional EIF2 initiation factor-mediated to alternative EIF2A-mediated uORF translation. These observations suggest that uORFs constitute an exciting new frontier in the field of translational regulation with the potential to fundamentally impact cellular fate.
Here, I propose to systematically analyze the function of uORFs during tumorigenesis. First, we will conduct an in vivo CRISPR/CAS9-based screen in mice to elucidate the role of thousands of uORFs in development, differentiation and upon oncogenic transformation. Second, focusing on select uORFs surfacing in the screen, we will document their role during tumor initiation and progression. Third, we will develop novel tools to detect uORF translation in vivo, exploit them to monitor uORF translation during different stages of tumorigenesis, gain mechanistic insight into their function and finally test the relevance of these findings in human cancer. Collectively, these approaches will provide unprecedented and comprehensive insight into the function of uORFs, unravel new paradigms in the control of gene expression and expose novel strategies for cancer diagnostics and treatment.
We recently unveiled a hitherto unappreciated role for upstream open reading frame (uORF) translation in tumorigenesis and unearthed a novel switch from conventional EIF2 initiation factor-mediated to alternative EIF2A-mediated uORF translation. These observations suggest that uORFs constitute an exciting new frontier in the field of translational regulation with the potential to fundamentally impact cellular fate.
Here, I propose to systematically analyze the function of uORFs during tumorigenesis. First, we will conduct an in vivo CRISPR/CAS9-based screen in mice to elucidate the role of thousands of uORFs in development, differentiation and upon oncogenic transformation. Second, focusing on select uORFs surfacing in the screen, we will document their role during tumor initiation and progression. Third, we will develop novel tools to detect uORF translation in vivo, exploit them to monitor uORF translation during different stages of tumorigenesis, gain mechanistic insight into their function and finally test the relevance of these findings in human cancer. Collectively, these approaches will provide unprecedented and comprehensive insight into the function of uORFs, unravel new paradigms in the control of gene expression and expose novel strategies for cancer diagnostics and treatment.
In Aim 1, we are carrying out an in vivo CRISPR/CAS9 screen to unveil the role of upstream open reading frames (uORFs) in tumorigenesis. At the time of this midterm report, we have set up a final version of the bioinformatics pipeline to annotate the exact translated genome-wide uORF genomic locations. This pipeline based on the program ORF-RATER uses our previous ribosome profiling data from wild-type skin, SOX2-positive tumor-initiating cells and squamous cell carcinomas to successfully map hundreds of uORFs, but also N-terminal elongation, N-terminal truncations as well as novel ORFs that are specifically expressed in cancer cells. This final list of candidate uORFs and their exact translated genomic locations were used to design individual sgRNAs.
We have then built sgRNA libraries to target these annotated uORFs in vivo. To this end, we were injecting the sgRNA library into E9.5 embryos by exploiting our intraamniotic in utero microinjection system. This system has been purchased, fully set up and up, also optimized in terms of high-titer lentivirus production and is now up and running at our mouse facility. Using this technique, we can successfully infect E9.5 embryos and are assessing the representation of this library postnatally in the different epidermal cell populations of wild-type and oncogenic animals.
One of the major limitations of pooled CRISPR screenings is the restriction to a simple readout such as proliferation. We have now further refined the CRISPR screening strategy and have developed a single-cell CRISPR platform to tackle the biology of uORFs in vivo. The strategy is based on the CROP-seq technique and has been further adjusted to meet our in vivo microinjection strategy. We have optimized this strategy o which enabled us now to test the role uORFs at single-cell resolution in different cell populations of the epidermis and allows us to link genetic perturbations to its full transcriptomic readout.
Using this advanced platform, we then cloned the sgRNA library into an adjusted CROP-seq plasmid, prepared high titer lentivirus and performed the systematic CRISPR screen to uncover the role of uORFs in tumorigenesis (Aim 1). In parallel, we finalized the advanced bioinformatics pipeline to analyze the single-cell CRISPR screening results. We have finalized the screen for two time points, P4 and P60 and have confirmed the top hits in a secondary screen. The validated top uORF candidates are currently in-depth characterized for their role in tumorigenesis.
We have then built sgRNA libraries to target these annotated uORFs in vivo. To this end, we were injecting the sgRNA library into E9.5 embryos by exploiting our intraamniotic in utero microinjection system. This system has been purchased, fully set up and up, also optimized in terms of high-titer lentivirus production and is now up and running at our mouse facility. Using this technique, we can successfully infect E9.5 embryos and are assessing the representation of this library postnatally in the different epidermal cell populations of wild-type and oncogenic animals.
One of the major limitations of pooled CRISPR screenings is the restriction to a simple readout such as proliferation. We have now further refined the CRISPR screening strategy and have developed a single-cell CRISPR platform to tackle the biology of uORFs in vivo. The strategy is based on the CROP-seq technique and has been further adjusted to meet our in vivo microinjection strategy. We have optimized this strategy o which enabled us now to test the role uORFs at single-cell resolution in different cell populations of the epidermis and allows us to link genetic perturbations to its full transcriptomic readout.
Using this advanced platform, we then cloned the sgRNA library into an adjusted CROP-seq plasmid, prepared high titer lentivirus and performed the systematic CRISPR screen to uncover the role of uORFs in tumorigenesis (Aim 1). In parallel, we finalized the advanced bioinformatics pipeline to analyze the single-cell CRISPR screening results. We have finalized the screen for two time points, P4 and P60 and have confirmed the top hits in a secondary screen. The validated top uORF candidates are currently in-depth characterized for their role in tumorigenesis.
We expect to systematically unravel the role of 5'UTRs in cancer, provide comprehensive insights into a novel layer within gene expression regulation and identify new targets for cancer therapy.