Periodic Reporting for period 5 - TRANSREG (Dissecting the role of Translational Regulation in Tumorigenesis)
Periodo di rendicontazione: 2024-05-01 al 2025-10-31
A central challenge in cancer biology is understanding how cells transition from normal homeostasis to malignancy, a process that cannot be explained by genetic mutations alone. Because mRNA abundance often poorly predicts protein levels in cancer, translational regulation has emerged as a critical yet underexplored determinant of cellular state and tumor evolution.
This project addressed the fundamental question of how altered protein synthesis programs contribute to clonal expansion, tumor initiation and malignant progression, processes central for the development of novel therapies improving cancer diagnosis and treatment.
Building on our initial discovery that upstream open reading frames and alternative translation initiation pathways play an unexpected role in tumorigenesis, the overall objective of this project was to systematically dissect gene regulatory pathways, including regulatory sequences and translational control mechanisms, in vivo. To this end, we developed and applied an in vivo single-cell CRISPR screening framework that enabled tissue-wide functional interrogation of cancer-relevant genes and upstream regulatory elements at single-cell resolution. Finally, we mechanistically characterized the role of newly identified regulators during tumor initiation and progression. Collectively, this project established new conceptual and technological foundations for studying clonal evolution and gene expression regulation in vivo, exposing novel entry points for cancer prevention and therapy.
This project addressed the fundamental question of how altered protein synthesis programs contribute to clonal expansion, tumor initiation and malignant progression, processes central for the development of novel therapies improving cancer diagnosis and treatment.
Building on our initial discovery that upstream open reading frames and alternative translation initiation pathways play an unexpected role in tumorigenesis, the overall objective of this project was to systematically dissect gene regulatory pathways, including regulatory sequences and translational control mechanisms, in vivo. To this end, we developed and applied an in vivo single-cell CRISPR screening framework that enabled tissue-wide functional interrogation of cancer-relevant genes and upstream regulatory elements at single-cell resolution. Finally, we mechanistically characterized the role of newly identified regulators during tumor initiation and progression. Collectively, this project established new conceptual and technological foundations for studying clonal evolution and gene expression regulation in vivo, exposing novel entry points for cancer prevention and therapy.
Malignant transformation is a multistep process that begins long before overt tumor formation, with randomly arising driver mutations giving rise to clonal expansions in phenotypically normal tissues. While such clonal expansions can remodel entire tissues, the mechanisms that determine why only a small subset of clones ultimately progress to malignancy remain poorly understood. Addressing this fundamental question is essential for improving cancer prevention, early detection and therapeutic intervention.
In this ERC project, we developed and applied an in vivo single-cell CRISPR screening approach to systematically investigate gene expression regulatory pathways, upstream open reading frames and translational control mechanisms directly within intact mammalian tissues. By combining ultrasound-guided in utero lentiviral delivery and an in vivo single-cell CRISPR strategy, we enabled longitudinal, tissue-wide monitoring of clonal expansions and the underlying gene expression programs at single-cell resolution in the mouse epidermis. Using this approach, we identified new gene programs that drive clonal growth in normal tissues and uncovered how these programs are rewired during tumor initiation and progression. In particular, we revealed context-dependent signalling programs and cell-cell interactions that promote clonal expansion in premalignant settings and are subsequently altered during malignant transformation, leading to invasive cancer cell states associated with poor clinical outcomes.
Using this platform, we specifically interrogated previously underexplored layers of gene regulation, including upstream open reading frames and translational control mechanisms. Systematic perturbation of these elements uncovered both global and cell-type-specific functions during tissue homeostasis and disease and demonstrated that disruption of select upstream regulatory sequences can strongly impact cellular proliferation and the translational landscape. These findings support the notion that translational regulation contributes to clonal behavior and tumor evolution, beyond transcriptional control alone.
Collectively, this ERC project establishes in vivo single-cell CRISPR screening as a powerful approach to study clonal evolution, gene regulatory networks and translational control in mammalian tissues. The published findings generated considerable interest and were widely covered by local and international press, underscoring their broader relevance. By integrating functional genomics with single-cell analyses, the project provides a basis for our current efforts aimed at understanding how altered gene regulation contributes to cancer progression and for developing improved diagnostic and therapeutic strategies.
In this ERC project, we developed and applied an in vivo single-cell CRISPR screening approach to systematically investigate gene expression regulatory pathways, upstream open reading frames and translational control mechanisms directly within intact mammalian tissues. By combining ultrasound-guided in utero lentiviral delivery and an in vivo single-cell CRISPR strategy, we enabled longitudinal, tissue-wide monitoring of clonal expansions and the underlying gene expression programs at single-cell resolution in the mouse epidermis. Using this approach, we identified new gene programs that drive clonal growth in normal tissues and uncovered how these programs are rewired during tumor initiation and progression. In particular, we revealed context-dependent signalling programs and cell-cell interactions that promote clonal expansion in premalignant settings and are subsequently altered during malignant transformation, leading to invasive cancer cell states associated with poor clinical outcomes.
Using this platform, we specifically interrogated previously underexplored layers of gene regulation, including upstream open reading frames and translational control mechanisms. Systematic perturbation of these elements uncovered both global and cell-type-specific functions during tissue homeostasis and disease and demonstrated that disruption of select upstream regulatory sequences can strongly impact cellular proliferation and the translational landscape. These findings support the notion that translational regulation contributes to clonal behavior and tumor evolution, beyond transcriptional control alone.
Collectively, this ERC project establishes in vivo single-cell CRISPR screening as a powerful approach to study clonal evolution, gene regulatory networks and translational control in mammalian tissues. The published findings generated considerable interest and were widely covered by local and international press, underscoring their broader relevance. By integrating functional genomics with single-cell analyses, the project provides a basis for our current efforts aimed at understanding how altered gene regulation contributes to cancer progression and for developing improved diagnostic and therapeutic strategies.
This project advanced beyond the state of the art by establishing and applying an in vivo single-cell CRISPR screening approach that enables tissue-wide, cell-type-resolved functional interrogation of genes and regulatory elements directly in intact mammalian tissues. Prior to this work, functional single-cell genomic screens were largely restricted to in vitro systems or bulk in vivo readouts, limiting the ability to link gene perturbations to cell-type-specific phenotypes within complex tissues. By integrating ultrasound-guided in utero lentiviral delivery with single-cell analyses and guide capture, this project enabled longitudinal analysis of clonal dynamics and gene regulatory programs at single-cell resolution in vivo.