Periodic Reporting for period 1 - OncoResist (Genetic and epigenetic resistance to oncogenic transformation of an epithelium)
Reporting period: 2021-01-01 to 2022-12-31
Oncogenesis is a complex multi-step process involving genetic and epigenetic alterations for normal cells to turn into cancerous cells. Remarkably, while some strong oncogenic signals can promote transformation in some cell states, they are entirely innocuous to other cell types.
My aim is to study this poorly understood phenomenon, being my current hypothesis, that intrinsic cell state transformation protection mechanisms are likely to exist.
I will use the Drosophila eye primordium epithelium, as I have previously observed that whereas committed epithelial cells over-proliferate in response to abnormal Hippo signalling, undifferentiated progenitor cells are resistant to this transformation.
To molecularly define such resilience to oncogenesis, the gene expression and chromatin accessibility profile of these two cell populations were compared. My preliminary results showed that the chromatin accessibility profile differed significantly and suggested that some pioneer transcription factors primed cells to respond to Hippo signalling. Moreover, I observed that non-coding RNAs were differentially expressed between these two cell populations. In particular, some long non-coding RNAs (lncRNAs), a class of molecules recently described as regulators of oncogenic signals, were particularly enriched in the progenitor cell population.
More specifically, I aim to address two main questions:
1 - Is the nuclear landscape (chromatin accessibility and TFs combinations) important to control the response to high oncogenic signals? And how much of this response relies on the activity of pioneer transcription factors?
2 - What is the role of the lncRNAs in controlling the cellular response to Hippo signalling?
A better understanding of the intrinsic cell state protective mechanisms against transformation is likely to open important new therapeutic approaches for cancer.
My aim is to study this poorly understood phenomenon, being my current hypothesis, that intrinsic cell state transformation protection mechanisms are likely to exist.
I will use the Drosophila eye primordium epithelium, as I have previously observed that whereas committed epithelial cells over-proliferate in response to abnormal Hippo signalling, undifferentiated progenitor cells are resistant to this transformation.
To molecularly define such resilience to oncogenesis, the gene expression and chromatin accessibility profile of these two cell populations were compared. My preliminary results showed that the chromatin accessibility profile differed significantly and suggested that some pioneer transcription factors primed cells to respond to Hippo signalling. Moreover, I observed that non-coding RNAs were differentially expressed between these two cell populations. In particular, some long non-coding RNAs (lncRNAs), a class of molecules recently described as regulators of oncogenic signals, were particularly enriched in the progenitor cell population.
More specifically, I aim to address two main questions:
1 - Is the nuclear landscape (chromatin accessibility and TFs combinations) important to control the response to high oncogenic signals? And how much of this response relies on the activity of pioneer transcription factors?
2 - What is the role of the lncRNAs in controlling the cellular response to Hippo signalling?
A better understanding of the intrinsic cell state protective mechanisms against transformation is likely to open important new therapeutic approaches for cancer.
The main objective of this project was to understand in vivo why high oncogenic signals promote cellular transformation in some cell states while other cell populations in the same tissue are resilient to transformation. To this end, we focused on studying the cellular response to the oncogene Yki in a model system with well-defined coordination of the transition between progenitor and differentiated states, the fruit fly D. melanogaster compound eye.
By a combination of genomic and genetic tools, we identified several players that might be modulating the nuclear landscape and potentially be underlying this differential response. We found a good correlation between the chromatin opening by pioneer factors and the activation of target genes by the Hippo signalling effector Yki. This correlation seems to be essential as changing the levels of pioneer factors affects the capacity of over-active Yki to induce uncontrolled proliferation and subsequently hyperplasia. Importantly, this regulatory mechanism might be evolutionarily conserved in higher eukaryotes as the pioneer factors orthologues have been shown to have a similar pioneer function and are frequently found to be amplified in cancers of epithelial origin.
This project could be divided into two main parts for the same objective which was to characterize how the different players regulating the nuclear landscape contribute to creating a permissive environment for the action of an oncogenic signal.
In the first part, we identified and characterized the contribution of the TFs and chromatin remodellers and in the second part we aimed to identify the contribution of lncRNAs to the regulation of the nuclear landscape. Due to the time limitations, we focused more on achieving the milestones and deliverables of the first part (Milestone 1 – Fully characterization of pioneer factors requirement for Yki-induced growth and Deliverable 2 – Final list of genes that affect Yki-induced overgrowth) and performing the technical optimization for the milestones of the second part that will be fully completed in my recently established lab during 2023 (Milestone 2 – Identification of the lncRNAs with impact on cell transformation, Milestone 3 – Establishment of the lncRNA action mechanism). Part of this identification was already performed as our TaDa data identified ncRNAs with differential expression and we are currently establishing mutants for those players to study their impact on growth control. It is important to highlight that the achievement of the first milestone generated the foundations for a manuscript that will be submitted in the next few months that will be made freely accessible to the scientific community.
Besides the identification of the synergy between pioneer factors and Yki, we also found novel players controlling Yki-induced overgrowth. Together with an MSc student under my direct supervision, we performed a systematic analysis to determine the impact of the different chromatin remodellers on Yki-induced overgrowth, resulting in a new deliverable – List of the chromatin remodellers affecting Yki activity and we are preparing a second paper to be submitted before the end of 2023.
These results are being exploited in two ways, the characterization of the nuclear landscape was the basis of my successful application to an independent Assistant Researcher to develop my independent research programme on the subject of growth control and oncogenesis, while Laura Castro is currently applying for PhD fellowships to explore the deliverables resulted from this project. Moreover, we are setting up novel connections with local Hospitals to explore the prevalence of pioneer factors mutations in cancers of epithelial origin in the Portuguese population.
By a combination of genomic and genetic tools, we identified several players that might be modulating the nuclear landscape and potentially be underlying this differential response. We found a good correlation between the chromatin opening by pioneer factors and the activation of target genes by the Hippo signalling effector Yki. This correlation seems to be essential as changing the levels of pioneer factors affects the capacity of over-active Yki to induce uncontrolled proliferation and subsequently hyperplasia. Importantly, this regulatory mechanism might be evolutionarily conserved in higher eukaryotes as the pioneer factors orthologues have been shown to have a similar pioneer function and are frequently found to be amplified in cancers of epithelial origin.
This project could be divided into two main parts for the same objective which was to characterize how the different players regulating the nuclear landscape contribute to creating a permissive environment for the action of an oncogenic signal.
In the first part, we identified and characterized the contribution of the TFs and chromatin remodellers and in the second part we aimed to identify the contribution of lncRNAs to the regulation of the nuclear landscape. Due to the time limitations, we focused more on achieving the milestones and deliverables of the first part (Milestone 1 – Fully characterization of pioneer factors requirement for Yki-induced growth and Deliverable 2 – Final list of genes that affect Yki-induced overgrowth) and performing the technical optimization for the milestones of the second part that will be fully completed in my recently established lab during 2023 (Milestone 2 – Identification of the lncRNAs with impact on cell transformation, Milestone 3 – Establishment of the lncRNA action mechanism). Part of this identification was already performed as our TaDa data identified ncRNAs with differential expression and we are currently establishing mutants for those players to study their impact on growth control. It is important to highlight that the achievement of the first milestone generated the foundations for a manuscript that will be submitted in the next few months that will be made freely accessible to the scientific community.
Besides the identification of the synergy between pioneer factors and Yki, we also found novel players controlling Yki-induced overgrowth. Together with an MSc student under my direct supervision, we performed a systematic analysis to determine the impact of the different chromatin remodellers on Yki-induced overgrowth, resulting in a new deliverable – List of the chromatin remodellers affecting Yki activity and we are preparing a second paper to be submitted before the end of 2023.
These results are being exploited in two ways, the characterization of the nuclear landscape was the basis of my successful application to an independent Assistant Researcher to develop my independent research programme on the subject of growth control and oncogenesis, while Laura Castro is currently applying for PhD fellowships to explore the deliverables resulted from this project. Moreover, we are setting up novel connections with local Hospitals to explore the prevalence of pioneer factors mutations in cancers of epithelial origin in the Portuguese population.
Besides the fact that there are reports showing the relationship between chromatin remodelers and Hippo signalling, the results from our project propose that there is a synergy between the activity of pioneer factors in opening the chromatin and the cellular response to the oncogenic form of the activated effector of the pathway, Yki. Interestingly, pioneer factors are highly versatile proteins with different targets according to the developmental context. Thus, understanding how their activity could be modulated might provide new clues to explain why cells respond differently to oncogenic signals. Moreover, it is our ultimate goal to understand the cell-intrinsic capacities to resist oncogenic signals to try to prevent the proliferation of transformed cells and develop new therapeutic strategies to drug new targets for cancer treatment.