Periodic Reporting for period 1 - BRCAstem (Monitoring cancer stem cell dynamics and therapeutic response in BRCA2-deficient breast tumour cells)
Período documentado: 2020-07-01 hasta 2022-06-30
Mutations in the breast cancer susceptibility genes (BRCA) 1 and 2 lead to an increased susceptibility to breast, ovarian, and other types of cancers. In recent years, PARP inhibitors rapidly transformed the treatment of such cancers. As an anti-cancer drug, these inhibitors stop the Poly-ADP Ribose Polymerase (PARP) protein from doing its repair work in cancer cells, leading to cell death. Normally, the BRCA1 and BRCA2 genes play an important role in cell repair (through a process called homologous repair, HR) so that cells are less likely to undergo successful DNA repair if there is a fault in one or both of these genes. In other terms, BRCA1/2-deficient cancer cells already have a compromised DNA repair system, therefore blocking PARP leads to the accumulation of DNA lesions and ultimately to the cancer cells’ death. However, it has been reported that tumours develop resistance to PARP inhibitors, which often entails the emergence of mutations that trigger rewiring of the damage response pathways within the tumour, so that cell death responses to treatment are replaced by cancer cell survival and metastasis. It has also been suggested that resistance can be linked to the propagation of rare, stem-like cells (also known as cancer-initiating or cancer stem cells), responsible for the sustained and uncontrolled growth of malignant tumours and proposed to play significant roles in metastasis and cancer recurrence in DNA repair-deficiency contexts.
In the current project, we propose to study the relationship between the propagation of stem-like cancer cells and the response to PARP in cancer cell lines and tumours models lacking BRCA1/2. The proposed project is based on a combination of interdisciplinary experimental and analytical strategies, at the interface between Molecular Biology (experimental setup and validation), Computational Biology (custom designed analytical tools) and Molecular Medicine (biomarker identification, treatment benchmarking and evaluation). Indeed, our goal is to understand the molecular mechanisms driving the emergence and dynamics of stem-like cells in models of BRCA2 inactivation, but also to evaluate the stem-like cell subpopulations’ impact on the response of BRCA-mutated tumours to current targeting therapies. Our novel approach combining histology information and gene expression analysis will allow to understand both which cell subpopulations are present (before and after treatments) as well as the physical interactions between them, bringing the analysis of intra-tumour heterogeneity to an unprecedented resolution level. Importantly, this work will help design novel biomarkers to stratify patients likely to respond to therapy and thus propose personalized treatment strategies. A gene expression signature indicative of specific cell dynamics within HR-defective tumours may provide new molecular targets for prognosis and treatment, allowing to predict and monitor therapeutic responses in BRCA1/2-deficient breast tumours. Thus, the proposed project is highly translational, directly aiming at improving therapy outcomes for patients with BRCA1/2 and other HR mutations.
In the current project, we propose to study the relationship between the propagation of stem-like cancer cells and the response to PARP in cancer cell lines and tumours models lacking BRCA1/2. The proposed project is based on a combination of interdisciplinary experimental and analytical strategies, at the interface between Molecular Biology (experimental setup and validation), Computational Biology (custom designed analytical tools) and Molecular Medicine (biomarker identification, treatment benchmarking and evaluation). Indeed, our goal is to understand the molecular mechanisms driving the emergence and dynamics of stem-like cells in models of BRCA2 inactivation, but also to evaluate the stem-like cell subpopulations’ impact on the response of BRCA-mutated tumours to current targeting therapies. Our novel approach combining histology information and gene expression analysis will allow to understand both which cell subpopulations are present (before and after treatments) as well as the physical interactions between them, bringing the analysis of intra-tumour heterogeneity to an unprecedented resolution level. Importantly, this work will help design novel biomarkers to stratify patients likely to respond to therapy and thus propose personalized treatment strategies. A gene expression signature indicative of specific cell dynamics within HR-defective tumours may provide new molecular targets for prognosis and treatment, allowing to predict and monitor therapeutic responses in BRCA1/2-deficient breast tumours. Thus, the proposed project is highly translational, directly aiming at improving therapy outcomes for patients with BRCA1/2 and other HR mutations.
We have sequenced, at single-cell resolution, the RNA of BRCA2-proficient and -deficient (28 days after BRCA2 expression depletion) cells to study gene expression patterns in both the normal context of BRCA2 expression and in the repair-deficiency context. This has allowed to confirm the presence of a cancer stem cell like subpopulation of cells, as well as to identify other gene expression signatures such as an enhanced immune response in BRCA2-deficient single-cells and the re-expression of BRCA2 in a subgroup of cells that were initially BRCA2-deficient. In addition, we have recently grown cell cultures of BRCA2-deficient cells into PARP inhibitor (PARPi) resistance and performed sequencing as afore-mentioned. This will allow us to compare gene expression patterns in treatment-naïve and PARPi-resistant cells. Finally, using an optimized protocol to dissociate and harvest tumour cells, we have sequenced patient-derived xenografts (PDX) tumour models that were either treatment-naïve or PARPi resistant, aiming to recapitulate the results obtained using cell cultures.
Notably, we are reporting the generation of gene expression maps, at single-cell resolution, of BRCA2-deficient cell lines and PDX tumour models, obtained by performing the state-of-the-art technique of single-cell RNA sequencing (scRNA-seq). Deeper analysis of highly expressed genes in each of the subpopulations identified will be studied for potential maker use. Indeed, the proposed project has a clear high-reward potential, as we expect it to lead to the successful identification of novel biomarkers as companion diagnostics for aggressive breast and ovarian cancers. In addition, this early-stage work will result in high-resolution spatial maps of physical interactions between cell subpopulations within BRCA2-deficient tumours, that will be made publicly available as curated repositories. This will warrant scientific transparency and will increase the visibility of the project. Furthermore, the data that will be made available might contribute to allow future larger-scale projects focusing on individual profiling based on patient-derived biopsies, allowing to better account for heterogeneity between patients and ultimately, test the robustness of the identified predictive biomarkers. As this project focuses on unknown mechanisms of drug failure, it is highly relevant to the field of translational breast cancer research and could lead to novel biomarkers and personalized treatment strategies.