INSTITUCIONES PARA EL DEPÓSITO DE VALORES

The use of preclinical mouse models in cancer has enabled researchers to learn about tumour biology in complicated and dynamic physiological systems. Their use is nowadays the most rate-limiting step in the translation of basic tumour biology discoveries into diagnostic and clinical applications. A great effort is being performed to obtain reliable cancer mouse models, which could truly mimic the natural course of human tumour initiation, growth and metastatic dissemination. This would allow to substantially advance translational cancer research216_.

With the aim of studying the in vivo causal contribution of the candidate PGC1α, we used a well-established PCa mouse model based on the deletion of the tumour suppressor PTEN in the prostate epithelium. PTEN expression is reduced in 69% of human PCa, and 86% of castration resistant prostate cancer (CRPC) metastatic patients58,217_{. The targeted deletion of Pten in genetic}

engineered mouse models (GEMMs) has been widely used in different studies to study PCa initiation and progression61,218_{. Furthermore, the implication of PTEN and its main downstream pathway, PI3K,}

has been studied in PCa pathogenesis and progression62,43_{. It is important to highlight that this model}

does not present signs of clinical metastasis62_{. As bone metastasis causes a significant clinical}

burden for PCa patients, it is nowadays the focus of PCa research. In fact, the survival rate of PCa

patients with metastatic disease has remained to be 30%

(https://seer.cancer.gov/statfacts/html/prost.html), due to the lack of curative therapeutics for advanced and metastatic disease. Nonetheless, models that mimic the widespread clinical phenomenon of bone metastasis in advanced PCa patients are scarce.

In this thesis work, we have demonstrated that the conditional deletion of Pgc1a and Pten in the prostate epithelium leads to clinical metastatic signs in lymph nodes and liver, and the presence of disseminated prostatic epithelial cells in the bone. Remarkably, two studies based on Pten deletion revealed metastatic signs both in the lymph nodes219 _{and in the pulmonary alveolar space}218_.

Furthermore, other models of metastasis based on autonomic nerve development163 _{and the}

concomitant deletion of Pten and the tumour suppressors´ p53 and SMAD4, have been characterised164,166_{. Yet, none of these models presented bone metastasis. In this regard, only the}

transgenic adenocarcinoma of the mouse prostate model (TRAMP), which is based on targeting the SV40 early region comprising the large T and small t antigens, progresses to pulmonary, lymph node and bone metastasis. Importantly, this mouse model is based on non-representative features of the disease220_{. Therefore, though the Pten deletion-based model may be more suitable for therapeutic}

studies to attenuate disease progression in the primary tumour, other models such as TRAMP could be used to analyse mechanisms of metastasis in vivo. This fact suggests that suitable animal tumour

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models should be developed to adequately mimic the evolution, the growth kinetics, the molecular and functional heterogeneity, and the phenotypic properties of human tumours. Due to the increasing knowledge developed in the last decade about tumour progression and dissemination, there is a great need to develop models to dissect the multistep pathways of tumorigenesis, which cannot be ascertained using cancer cell lines in culture nor human specimens.

The mouse model used in this thesis work is a second/third generation mouse model, as it allows the conditional and spatially controlled deletion of a tumour suppressor and metabolic co- regulator. Of note, orthotopic xenotransplants (considered first generation models) are highly used to study the mechanisms leading to cancer progression and metastasis216_{. Therefore, the use of these}

techniques could improve the understanding of PGC1α function in metastasis initiation in our work. Moreover, other models are being generated based on patient-derived xenografts and spontaneous tumours (fourth generation models) and on high-throughput technologies such as CRISPR/Cas9, that allow a faster functional testing of candidate genes. These new techniques will allow the study and expression modulation of target genes in a time and space controlled manner. Regardless the limitations of our model, timing is also a key factor. Due to technical and ethical facts, mice could not be maintained for longer time, which could have given us some clues about metastasis initiation and progression. Furthermore, and according to the invasive signs observed, it would be very interesting to analyse younger (3 months of age) DKO mice, to see whether Pgc1α loss affects not only progression, but also the initiation of cancer in vivo, as we have been able to see in vitro using PCa cell lines.

To our knowledge, this is the first insight of enhanced progression of PCa based on the deletion of a principal regulator of metabolism (PGC1α) upon Pten loss. Moreover, although the implication of PGC1α in systemic metabolism has been extensively investigated 174,221,222_{, its activity}

in cancer is beginning to be understood. Interestingly, the loss of PGC1α in vivo protects against carcinogenesis, impairing tumour growth through mitochondrial and fatty acid metabolism regulation112_{. In contrast, the results from our in vivo model support the notion that PGC1α acts as a}

tumour suppressor in PCa. In line with our results, PGC1α depletion led to a higher number of intestinal tumours in mice, exerting a pro-apoptotic activity and tumour suppressor role113_{. Of note,}

different tissue types show profound differences in tumorigenesis, organization of oncogenic signalling pathways, and in their response to oncogenic driver mutations57_{. This could be one of the}

main obstacles when studying tumour development and evolution. The relevance of finding the precise experimental systems will be essential to ascertain the non-answered questions in cancer research. Of note, a big effort has been made during the last decades aiming to generate 3D organotypic cultures. These are derived from primary tissues, pluripotent stem cells, established cell lines, and whole or segmented organs, which consist of multiple tissue types223_{. Strikingly, organoid}

lines derived from PCa patients recapitulate the molecular diversity of PCa subtypes and maintain tumour identity, making them amenable to genetic and pharmacologic studies. In summary, this new

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line of techniques has enabled a high reproducibility of in vivo systems in cancer to test drug response and the design of personalized therapy224,225_.