Tuberous Sclerosis Complex 1 Regulates dE2F1 Expression during Development and Cooperates with RBF1 to Control Proliferation and Survival

Previous studies in Drosophila melanogaster have demonstrated that many tumor suppressor pathways impinge on Rb/E2F to regulate proliferation and survival. Here, we report that Tuberous Sclerosis Complex 1 (TSC1), a well-established tumor suppressor that regulates cell size, is an important regulator of dE2F1 during development. In eye imaginal discs, the loss of tsc1 cooperates with rbf1 mutations to promote ectopic S-phase and cell death. This cooperative effect between tsc1 and rbf1 mutations can be explained, at least in part, by the observation that TSC1 post-transcriptionally regulates dE2F1 expression. Clonal analysis revealed that the protein level of dE2F1 is increased in tsc1 or tsc2 mutant cells and conversely decreased in rheb or dTor mutant cells. Interestingly, while s6k mutations have no effect on dE2F1 expression in the wild-type background, S6k is absolutely required for the increase of dE2F1 expression in tsc2 mutant cells. The canonical TSC/Rheb/Tor/S6k pathway is also an important determinant of dE2F1-dependent cell death, since rheb or s6k mutations suppress the developmentally regulated cell death observed in rbf1 mutant eye discs. Our results provide evidence to suggest that dE2F1 is an important cell cycle regulator that translates the growth-promoting signal downstream of the TSC/Rheb/Tor/S6k pathway

LKB1 loss links serine metabolism to DNA methylation and tumorigenesis

Intermediary metabolism generates substrates for chromatin modification, enabling the potential coupling of metabolic and epigenetic states. Here we identify a network linking metabolic and epigenetic alterations that is central to oncogenic transformation downstream of the liver kinase B1 (LKB1, also known as STK11) tumour suppressor, an integrator of nutrient availability, metabolism and growth. By developing genetically engineered mouse models and primary pancreatic epithelial cells, and employing transcriptional, proteomics, and metabolic analyses, we find that oncogenic cooperation between LKB1 loss and KRAS activation is fuelled by pronounced mTOR-dependent induction of the serine-glycine-one-carbon pathway coupled to S-adenosylmethionine generation. At the same time, DNA methyltransferases are upregulated, leading to elevation in DNA methylation with particular enrichment at retrotransposon elements associated with their transcriptional silencing. Correspondingly, LKB1 deficiency sensitizes cells and tumours to inhibition of serine biosynthesis and DNA methylation. Thus, we define a hypermetabolic state that incites changes in the epigenetic landscape to support tumorigenic growth of LKB1-mutant cells, while resulting in potential therapeutic vulnerabilities

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

Context-Dependent Requirement for dE2F during Oncogenic Proliferation

The Hippo pathway negatively regulates the cell number in epithelial tissue. Upon its inactivation, an excess of cells is produced. These additional cells are generated from an increased rate of cell division, followed by inappropriate proliferation of cells that have failed to exit the cell cycle. We analyzed the consequence of inactivation of the entire E2F family of transcription factors in these two settings. In Drosophila, there is a single activator, dE2F1, and a single repressor, dE2F2, which act antagonistically to each other during development. While the loss of the activator dE2F1 results in a severe impairment in cell proliferation, this defect is rescued by the simultaneous loss of the repressor dE2F2, as cell proliferation occurs relatively normally in the absence of both dE2F proteins. We found that the combined inactivation of dE2F1 and dE2F2 had no significant effect on the increased rate of cell division of Hippo pathway mutant cells. In striking contrast, inappropriate proliferation of cells that failed to exit the cell cycle was efficiently blocked. Furthermore, our data suggest that such inappropriate proliferation was primarily dependent on the activator, de2f1, as loss of de2f2 was inconsequential. Consistently, Hippo pathway mutant cells had elevated E2F activity and induced dE2F1 expression at a point when wild-type cells normally exit the cell cycle. Thus, we uncovered a critical requirement for the dE2F family during inappropriate proliferation of Hippo pathway mutant cells

Functional Integration of the pRB and Hippo Pathways in Drosophila Prevents Inappropriate Proliferation.

Functional Integration of the pRB and Hippo Pathways in Drosophila Prevents Inappropriate Proliferation

Loss of <i>de2f1</i> does not block induction of cyclin E in <i>wts</i> mutant cells.

<p>Clones of mutant cells were generated with <i>ey</i>-FLP and distinguished by the lack of GFP (green). (A) In wild type eye imaginal discs, cyclin E (magenta) expression is elevated within and immediately posterior to the morphogenetic furrow (MF). In <i>wts</i> mutant cells (B) and in <i>de2f1 wts</i> double mutant cells (C) cyclin E is expressed further posterior. Position of MF is shown by arrowhead. Posterior is to the right.</p

Elevated E2F activity in cells with inactivated Hippo pathway.

<p>Clones of mutant cells were induced with <i>ey</i>-FLP. Position of the morphogenetic furrow (MF) is shown by arrowhead. Posterior is to the right. (A–C) Expression of the E2F reporter, <i>PCNA</i>-GFP, (red) in the wild type eye disc (A), or in the eye discs containing clones of <i>wts</i> mutant tissue (B) and <i>de2f1 wts</i> double mutant tissue (C). (A) In wild type disc, the E2F reporter is expressed in a narrow stripe (red) immediately posterior to the MF, preceding S phase entry into the second mitotic wave (SMW). (B) In <i>wts</i> mutant cells which are marked by the absence of β-Gal (green), the E2F reporter is inappropriately expressed in the posterior region of the eye disc. Mutant tissue is outlined. (C) Inappropriate expression of the E2F reporter in the posterior region of the eye disc is absent in <i>de2f1 wts</i> double mutant cells. Note, that clones of <i>wts de2f1</i> double mutant cells were marked with β-Gal (green) produced from the <i>de2f1⁷²⁹</i> mutant allele. <i>de2f1 wts</i> double mutant tissue is outlined. (D) Endogenous dE2F1 (magenta) is expressed within the MF in a wild type disc as revealed by anti-dE2F1 antibody. (E–G, I) Clones of mutant cells were induced with <i>ey</i>-FLP and mutant tissue is identified by the lack of GFP (green). (E) The anti-dE2F1 antibody is highly specific as the staining is absent in <i>de2f1</i> mutant tissue (lack of green in E and pointed by the arrow). (F) <i>wts</i> and <i>hpo</i> mutant cells have an increased level of dE2F1 within the MF and inappropriately express dE2F1 posterior to the MF. Examples are pointed by the arrows. Position of mutant tissue is outlined. (G) Expression of endogenous dE2F1 protein (magenta) is unaffected in <i>ago</i> mutant cells in larval imaginal eye discs. (H) cyclin E was expressed ectopically in wild type mitotic clones using the MARCM system. Ectopic expression of cyclin E fails to elevate level of dE2F1 protein (magenta) posterior to the MF. Cells that express cyclin E are marked with GFP (green) and are outlined. (I) Endogenous dE2F2 protein (red) is expressed ubiquitously throughout the eye disc. Level of dE2F2 protein remains the same in both <i>wts</i> mutant and wild type tissue. (J) <i>de2f1</i> is transcriptionally induced in <i>hpo</i> mutant cells as revealed by the <i>de2f1</i> enhancer trap allele, <i>de2f1⁷²⁹</i>. <i>de2f1⁷²⁹</i> contains the <i>lacZ</i> insertion into the endogenous <i>de2f1</i> gene. The <i>lacZ</i> expression reflects transcription from the <i>de2f1</i> promoter <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000205#pgen.1000205-Reis1" target="_blank">[34]</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000205#pgen.1000205-Brook1" target="_blank">[35]</a>. Staining with anti-β-Gal antibody (magenta) was used to reveal expression of the <i>lacZ</i> gene in <i>de2f1⁷²⁹</i>. (K) SL2 cells were treated with nonspecific (NS), dE2F1 (E1), Warts (Wts) and Hippo (Hpo) dsRNA to deplete the corresponding proteins by RNAi. Cell extracts were analyzed by Western blot using antibody specific for Wts, dE2F1 and dE2F2. Depletion of Wts and Hpo shows an increase in the level of dE2F1 protein. In contrast, the dE2F2 protein level is not affected. The same blots were re-probed with anti-Tubulin antibody to control for equal loading. (L) Endogenous E2F activity is elevated in Hpo or Wts depleted SL2 cells. SL2 cells were incubated with non-specific (NS), RBF1, Hpo, and Wts dsRNAs for 4 days to deplete the corresponding proteins. On day 4, the E2F reporter (<i>PCNA</i>-luc) was transfected into the depleted cells and the luciferase activity was measured 2 days later to determine the level of the endogenous E2F activity in these cells. The pIE-LacZ plasmid was co-transfected to normalize for transfection efficiency. Results depict the mean of three experiments. Unpaired Student's <i>t</i>-Test assuming equal variance concluded that the increase of <i>PCNA-</i>luc reporter activity in RBF1, Hpo and Wts depleted cells was statistically significant from the NS control. RBF1 and Hpo depleted cells had a p-Value <0.001. Wts depleted cells had a p-Value <0.03.</p

Stabilized dE2F1 can induce apoptosis through transcriptional and non-transcriptional mechanisms.

<p>(1) During S-phase, a dE2F1 protein without a PIP-degron but with an intact DNA–binding domain will promote apoptosis through transcriptional activation of pro-apoptotic genes, such as <i>hid</i>. (2) A dE2F1 protein without a PIP-degron and lacking an intact DNA-binding domain can promote apoptosis through an unknown mechanism that requires a physical interaction with the RBF1 protein; this unknown mechanism is also dependent upon <i>hid</i>.</p