Combined Inactivation of pRB and Hippo Pathways Induces Dedifferentiation in the Drosophila Retina

Functional inactivation of the Retinoblastoma (pRB) pathway is an early and obligatory event in tumorigenesis. The importance of pRB is usually explained by its ability to promote cell cycle exit. Here, we demonstrate that, independently of cell cycle exit control, in cooperation with the Hippo tumor suppressor pathway, pRB functions to maintain the terminally differentiated state. We show that mutations in the Hippo signaling pathway, wts or hpo, trigger widespread dedifferentiation of rbf mutant cells in the Drosophila eye. Initially, rbf wts or rbf hpo double mutant cells are morphologically indistinguishable from their wild-type counterparts as they properly differentiate into photoreceptors, form axonal projections, and express late neuronal markers. However, the double mutant cells cannot maintain their neuronal identity, dedifferentiate, and thus become uncommitted eye specific cells. Surprisingly, this dedifferentiation is fully independent of cell cycle exit defects and occurs even when inappropriate proliferation is fully blocked by a de2f1 mutation. Thus, our results reveal the novel involvement of the pRB pathway during the maintenance of a differentiated state and suggest that terminally differentiated Rb mutant cells are intrinsically prone to dedifferentiation, can be converted to progenitor cells, and thus contribute to cancer advancement

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

Cooperation between dE2F1 and Yki/Sd defines a distinct transcriptional program necessary to bypass cell cycle exit

The Hippo signaling pathway regulates organ size homeostasis, while its inactivation leads to severe hyperplasia in flies and mammals. The transcriptional coactivator Yorkie (Yki) mediates transcriptional output of the Hippo signaling. Yki lacks a DNA-binding domain and is recruited to its target promoters as a complex with DNA-binding proteins such as Scalloped (Sd). In spite of recent progress, an open question in the field is the mechanism through which the Yki/Sd transcriptional signature is defined. Here, we report that Yki/Sd synergizes with and requires the transcription factor dE2F1 to induce a specific transcriptional program necessary to bypass the cell cycle exit. We show that Yki/Sd and dE2F1 bind directly to the promoters of the Yki/Sd-dE2F1 shared target genes and activate their expression in a strong cooperative manner. Consistently, RBF, a negative regulator of dE2F1, negates this synergy and limits the overall level of expression of the Yki/Sd-dE2F1 target genes. Significantly, dE2F1 is needed for Yki/Sd-dependent full activation of these target genes, and a de2f1 mutation strongly blocks yki-induced proliferation in vivo. Thus, the Yki transcriptional program is determined through functional interactions with other transcription factors directly at target promoters. We suggest that such functional interactions would influence Yki activity and help diversify the transcriptional output of the Hippo pathway

Cooperation between dE2F1 and Yki/Sd defines a distinct transcriptional program necessary to bypass cell cycle exit

The Hippo signaling pathway regulates organ size homeostasis, while its inactivation leads to severe hyperplasia in flies and mammals. The transcriptional coactivator Yorkie (Yki) mediates transcriptional output of the Hippo signaling. Yki lacks a DNA-binding domain and is recruited to its target promoters as a complex with DNA-binding proteins such as Scalloped (Sd). In spite of recent progress, an open question in the field is the mechanism through which the Yki/Sd transcriptional signature is defined. Here, we report that Yki/Sd synergizes with and requires the transcription factor dE2F1 to induce a specific transcriptional program necessary to bypass the cell cycle exit. We show that Yki/Sd and dE2F1 bind directly to the promoters of the Yki/Sd-dE2F1 shared target genes and activate their expression in a strong cooperative manner. Consistently, RBF, a negative regulator of dE2F1, negates this synergy and limits the overall level of expression of the Yki/Sd-dE2F1 target genes. Significantly, dE2F1 is needed for Yki/Sd-dependent full activation of these target genes, and a de2f1 mutation strongly blocks yki-induced proliferation in vivo. Thus, the Yki transcriptional program is determined through functional interactions with other transcription factors directly at target promoters. We suggest that such functional interactions would influence Yki activity and help diversify the transcriptional output of the Hippo pathway