Tracking and Targeting Tumor States at single-cell resolution in real time in vivo

Project Information

TrackingTumorStates

Grant agreement ID: 885093

DOI

10.3030/885093

EC signature date 15 June 2020

Start date 1 November 2020

End date 31 October 2026

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 2 500 000,00

EU contribution

€ 2 500 000,00

Coordinated by

UNIVERSITE LIBRE DE BRUXELLES
Belgium

Periodic Reporting for period 3 - TrackingTumorStates (Tracking and Targeting Tumor States at single-cell resolution in real time in vivo)

Reporting period: 2023-11-01 to 2025-04-30

Summary of the context and overall objectives of the project

Defining tumour state identities and functions at single-cell resolution in real time in vivo
Not all cancer cells in a tumour are alike. Some cancer cells proliferate, others differentiate, migrate and metastasise. Yet others enter a dormant state and resist chemotherapy. Scientists therefore need to identify distinct tumour states (TSs) and the mechanisms regulating their identities and functions. The EU-funded TrackingTumorStates project aims to comprehensively define the identities and functions of distinct TSs at single-cell resolution in squamous cell carcinoma. It will develop new genetically engineered tumour models to visualise the dynamics of TSs in real time in vivo. It will also evaluate the roles of the identified TSs by lineage ablation and determine their regulating mechanisms. This work will help to identify new tumour vulnerabilities and pave the way for new therapies.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

To define intra-tumour heterogeneity and unravel the distinct TS present in primary tumours and metastases, we have performed single cell RNA-seq from skin squamous cell carcinoma (SCC) presenting EMT. We identified tumor cells and stromal cells consisting of cancer associated fibroblasts (CAFs), different immune cells (neutrophils, macrophages, T and B cells), endothelial cells and pericytes. Bioinformatic analysis of the different skin SCCs with EMT revealed the presence of different TS ranging from pure epithelial (Epcam+, Krt14+, Cdh1+, Vim-), early hybrid EMT (Epcam-, Krt14+, Vim+), late hybrid EMT (Epcam-, Krt14-, Vim+, KRT8+) and full EMT (Epcam-, Krt14-, Vim+, Aspn+).

We have studied the intrinsic regulator of different tumor states. FAT1 is among the most frequently mutated driver genes in a broad range of human cancers. Despite the high frequency of FAT1 mutations, its role in cancer was poorly understood. Using skin SCC as a model, we found that Fat1 deletion accelerated tumor initiation and malignant progression and promoted hybrid EMT phenotype. Fat1 deleted skin SCCs presented increased tumor stemness and spontaneous metastasis. We identified drug resistance and vulnerabilities in FAT1 deficient tumors with important implications for cancer therapy.

During the transition from benign tumors to malignant carcinoma, tumor cells need to repress differentiation state and acquire invasive features by promoting EMT state. The non-genetic mechanisms required to sustain malignant tumor state are poorly understood. We identified NR2F2 as uniquely expressed in malignant SCC. We demonstrate that NR2F2 promotes tumor cell proliferation, EMT and invasive features, while repressing tumor differentiation and immune cell infiltration, making NR2F2 an ideal target for drug-induced cancer differentiation.

It has been suggested that EMT plays a role in the acquisition of resistance to anti-cancer therapy. However, the mechanism by which cancer cells presenting EMT resist to anti-cancer therapy is currently unknown. Using gain and loss of function in vitro and in vivo, we uncovered that RhoJ, a small GTPase preferentially expressed in EMT cancer cells, controls resistance to therapy. We found that RhoJ regulates EMT associated resistance to chemotherapy by enhancing the response to replicative stress and activating the DNA damage response, allowing tumor cells to rapidly repair DNA lesions induced by chemotherapy.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

1. The demonstration that mutation in a single gene promote hybrid EMT in different epithelial cancers was not at all expected and clearly demonstrate that genetic mutations can promote the preferential occurrence of certain tumor states. Such finding is advancing the research field significantly beyond the state of the art. In addition, the identification of tumor vulnerabilities in Fat1 mutated tumors, which are extremely frequent mutations in human cancer, is also very exciting for the field. We have patented such discovery and hope that our discovery will lead to future clinical trials that will advance the progress of cancer therapy in Fat1 mutated cancers.
2. The demonstration that epigenetic upregulation of one gene is capable of dramatically alter the malignant tumor states was also not expected. The identification of a key regulator of malignant state is advancing the research field significantly beyond the state of the art. Deletion of NR2F2 induces tumor regression by promoting apoptosis and tumor differentiation as well as decreasing cell proliferation. Considering that NR2F2 is overexpressed in a wide range of human cancers makes NR2F2 an ideal candidate for drug development.
3. The identification that a small GTPase mediates the resistance of EMT tumor cells to chemotherapy was very interesting and not at all expected. The identification of the molecular mechanisms by which RhoJ controls resistance to chemotherapy by regulating nuclear action polymerization, which in turn promote the recruitment of new origin of DNA replication allowing the tumor cells to repair DNA faster was very surprising and is advancing the research field significantly beyond the state of the art, opening new strategies for the development of anti-cancer therapy.

During the second part of the project, we hope to define the specific role for each tumor states using lineage tracing, intravital imaging and lineage ablation. We also wish to define the transcription factors that regulate the different transition states and the define the role and importance of the communications between the different tumor states and the different stromal cells that compose the tumor microenvironment.