Retinoblastoma and Hippo pathways cooperate to regulate cell proliferation and maintain differentiation

A tremendous amount of functional integration among pathways ensures proper development and maintains tissue homeostasis. One such example is the cooperation between the Retinoblastoma (Rb) and the Hippo tumor suppressor pathways. We know that the crosstalk occurs on at least two levels: in the regulation of cellular proliferation and in maintenance of terminal differentiation. We uncovered a novel mode of mechanism, by which the Hippo pathway controls cell cycle exit in cooperation with the Rb pathway. In this work I demonstrate that the chromatin-binding protein GAGA Factor (GAF), is a novel and critical partner in transcriptional regulation by the downstream effectors of the Rb and Hippo pathways, Yki/Sd and dE2f1. I show that functional GAF and its binding to the promoters of target genes common to dE2f1-Yki/Sd is required for both developmental and ectopic cell proliferation. The differentiation defects following simultaneous inactivation of the Rb and Hippo pathways manifest themselves as the failure to maintain cell-specific markers. I created a genetic lineage-tracing tool to visualize the cells, which have lost their identity, with the expression of the lacZ gene. I optimized the conditions for single cell analysis to determine the mechanism by which Rb and Hippo pathways cooperatively guard the state of terminal differentiation. Initial analysis confirmed the success of our approach and identified mutant cells marked by lacZ. Single cell RNA sequencing will give insight into the precise transcriptional signature of the mutant and wild type cells. A new emerging point of crosstalk appears to be at the level of mitochondrial activity, which is evident from studies in both flies and mammals. In order to explore this further, I first asked whether therapeutic modulation of the Rb pathway in human breast cancer cells would influence mitochondrial activity. I show that accumulation of active pRb leads to an increase in mitochondria-associated gene expression. This, subsequently, elevates mitochondrial activity and oxidative phosphorylation. Interestingly, enhanced mitochondrial activity sensitizes the normal MCF10A and MCF7 cancer cells to apoptosis, but not the aggressive MDA-MB-231 cancer cells. The role of the Hippo pathway in this context remains to be determined.