Dissection of the role of MiT/TFE Protein Family members on the survival of Disseminated indolent Breast cancer cells

Late relapses of metastatic cancers are an important aspect in the evolution of some cancer types and understanding their mechanisms could potentially save millions of lives. Although the organ microenvironment plays a critical role, the mechanisms sustaining the survival of disseminated tumor cells (DTC) over long periods of clinical latency are still not completely elucidated. Our research group has developed and validated an in vitro lung organotypic system combining the culture of breast cancer cells together with lung epithelial cells mimicking the lung alveolar microenvironment. This organotypic system has allowed the discovery of SFRP2 and EPHB6 as regulators of Disseminated Dormant Cancer cells (DDCC) crosstalk with lung epithelial cells. The latter was shown to sustain lysosomal accumulation by acting on MIT/TFE proteins, master regulators of autophagy-lysosomal pathway involved in several diseases such as neurodegenerative pathologies (Alzheimer, Parkinson), Lysosomal Storage diseases (LSD), and cancer. To date, it is unclear which member of the MIT/TFE protein family supports the lysosomal accumulation mediating DDCCs survival in the organotypic system and in vivo. Understanding the genetic drivers of lysosomal accumulation will allow to dissect their requirement in greater detail in vitro and in vivo. Hence, we propose to attempt clarifying this grey area in this master thesis. To this end, we employed two independent approaches: first, a stable knock down of Tfeb and Tfe3 with CRISPR-Cas9 mediated genome editing and second, a transient knock down of the same proteins by siRNA-mediated RNA interference, in D2.0R cells, a model of breast cancer dormancy. After validation of the proteins knock down, we studied their effect on D2.0R in vitro and in vivo. In vitro we addressed the lysosomal response to TFEB and TFE3 loss of function, as well as the proliferation capacity of cells with Tfeb and Tfe3 knock down both in monoculture and in the coculture system. Furthermore, we exploited longitudinal imaging in mice to study the effect of Tfeb knockdown on the proliferation capacities of D2.0R cells in vivo. Our data have confirmed a role of TFEB and TFE3 in the lysosomal accumulation in DDCCs in the organotypic system. Regardless of the technique, Tfeb and Tfe3 knock down has resulted in a partial inhibition of the lysosomal pathway and a nonlinear response on cell proliferation. We explained this with the redundancy between TFE3 and TFEB, and a possible compensatory effect of MITF, other member of MIT/TFE family not yet addressed in our study.