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

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

Yki-driven proliferation of cells posterior to the SMW is blocked in the absence of the dE2F family.

<p>(A) The pattern of S phases in the wild type eye discs as revealed by BrdU labeling. Position of the morphogenetic furrow (MF) is shown by arrowhead. Posterior is to the right. Wild type cells asynchronously proliferate anterior to the MF, arrest in G1 in the MF and undergo a synchronous S phase in the second mitotic wave (SMW) posterior to the MF. (B–C) Clones of wild type (B) and <i>dDP</i> mutant (C) cells overexpressing <i>yki</i> were generated with the MARCM system and marked with GFP (green). Clones in (B) were generated with the <i>ey</i>-FLP while clones in (C) were generated with the <i>hs</i>-FLP. (B) Posterior to the SMW, wild type cells that overexpress <i>yki</i> fail to exit the cell cycle and proliferate inappropriately as evident by the appearance of BrdU positive cells. (C) In contrast, <i>yki</i> is unable to induce inappropriate proliferation of <i>dDP</i> mutant cells posterior to the SMW. Note, that <i>dDP</i> mutant cells that overexpress <i>yki</i> show a normal pattern of BrdU incorporation in the SMW but do not incorporate BrdU posterior to the SMW. (D–M) Clones of mutant cells of different genotypes were generated with <i>ey</i>-FLP and the mutant tissue was distinguished by the lack of GFP (green). (D–F) Mosaic larval eye discs were labeled with BrdU (red) to detect the S phases (D and E) or stained with anti-phosH3 (magenta) to visualize mitoses (F). (D) <i>wts</i> mutant cells fail to exit the cell cycle posterior to the SMW and undergo inappropriate proliferation, which is evident by the persistence of BrdU incorporation (pointed by arrows). (E–F) In contrast, inappropriate proliferation posterior to the SMW is strongly reduced in <i>de2f1 de2f2 wts</i> triple mutant cells as judged by the absence of cells in S phase (red in E) or in mitosis (magenta in F) (pointed by arrows). (G–H) BrdU incorporation (red) in 12 hr pupal eye discs. (G) <i>wts</i> mutant cells continue unscheduled proliferation during early pupal development while wild type cells remain fully quiescent as revealed by BrdU labeling. (H) Inappropriate BrdU incorporation is absent in clones of <i>de2f2 de2f1 wts</i> triple mutant cells (a clone is pointed by arrow). (I–M) Pupal retina at 48 hr APF stained with anti-Discs large protein (Dlg) (red) to visualize cell outlines. (I) Wild type retina contains a single layer of interommatidial cells between ommatidial clusters. (J) Inappropriate proliferation of <i>wts</i> mutant cells posterior to the SMW and resistance of these cells to the developmental apoptosis during the pupal stage gives rise to the dramatic excess of interommatidial cells (pointed by arrow). (K) In contrast, the number of interommatidial cells is significantly reduced in <i>de2f1 de2f2 wts</i> triple mutant tissue (indicated by arrows). (L–M) The MARCM technique was used to overexpress <i>yki</i> in wild type (L) or in <i>dDP</i> mutant cells (M). A <i>dDP</i> mutation dramatically reduces supernumerary interommatidial cells which arise when <i>yki</i> is expressed. (N) Quantification of the number of interommatidial cells in pupal retina shown in (J–M). Data were collected from 23 ommatidia clusters for wild type, 11 ommatidia clusters for <i>wts</i>, 24 ommatidia clusters for <i>de2f1 de2f2 wts</i>, 12 ommatidia clusters for <i>tub</i>><i>yki</i> and 17 ommatidia clusters for <i>tub</i>><i>yki</i> in <i>DP^−/−</i>. The following abbreviations were used: <i>e1 e2 wts</i> corresponds to <i>de2f1 de2f2 wts</i>; <i>yki</i> corresponds to <i>tub</i>><i>yki</i> and <i>yki</i> in <i>DP</i> corresponds to <i>tub</i>><i>yki</i> in <i>DP^−/−</i>. Error bars represent standard deviations. Note that in comparison to the wild type tissue there is a small excess of interommatidial cells in <i>de2f1 de2f2 wts</i> triple mutant tissue and in <i>dDP</i> mutant tissue that overexpresses <i>yki</i>. This is likely due to the failure to execute normal pupal developmental apoptosis in these cells.</p

Loss of <i>de2f1</i> does not affect resistance to DNA damage and pupal developmental apoptosis in <i>wts</i> mutants.

<p>In all panels, clones were generated with the <i>ey</i>-FLP/FRT technique and mutant tissue is distinguished by the absence of GFP (green). (A–B) The pupal eye discs at 30 hr APF containing clones of <i>wts</i> mutant (A) and <i>de2f1 wts</i> double mutant (B) cells were stained with anti-Cleaved Caspase3 (C3) antibody (red) to detect apoptotic cells. In the pupal eye discs, developmentally regulated apoptosis is abundant in wild type cells (green) but is largely absent in <i>wts</i> mutant tissue (lack of green) and in <i>de2f1 wts</i> double mutant tissue indicating that <i>de2f1 wts</i> double mutant cells, like <i>wts</i> mutant cells, are protected from the cell death. (C) DNA damage induced apoptosis following irradiation was detected with anti-C3 antibody (red). There is an extensive apoptosis in wild type tissue. In contrast, <i>de2f1 wts</i> double mutant cells are protected from apoptosis after DNA damage. An example of <i>de2f1 wts</i> double mutant tissue is pointed by arrow. The morphogenetic furrow is marked by the arrowhead.</p