Introduction {#s1}
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Clinically detectable cancer cells carry a multitude of mutations and chromosomal aberrations, and they display an enormous genetic heterogeneity ([@bib45]; [@bib59]; [@bib6]; [@bib34]; [@bib57]). It is therefore desirable to target earlier tumorigenic stages but we know comparatively little about how pre-cancerous cells progressively develop into tumors ([@bib36]). The model system *Drosophila* allows analyzing the behavior of cells lacking particular tumor suppressor functions. During the growth phase (larval instars), the cells of the imaginal discs (that will eventually give rise to adult appendages) remain diploid and proliferate until the discs have reached an appropriate size. The simple architecture of the imaginal discs (the disc proper consists of a single cell-layered epithelium and is covered by the peripodial epithelium) enables the labeling and tracking of cell populations. These cell populations can be genetically manipulated with the help of sophisticated tools. Finally, since the larvae live in their food, cellular stress situations can be imposed by controlling the food source.

We have focused our analysis on cells lacking the tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10). PTEN is well conserved from flies to humans, and it is the second most frequently mutated tumor suppressor found in many types of human cancers ([@bib18]; [@bib46]; [@bib23]; [@bib52]). PTEN antagonizes the function of the lipid kinase Phosphatidylinositide 3-kinase (PI3K); therefore, in the absence of PTEN*,* high levels of the lipid second messenger PIP3 result in an increased membrane recruitment and activation of the serine/threonine kinase PKB (protein kinase B, also known as Akt), which leads to enhanced cellular growth, proliferation, and survival ([@bib2]; [@bib16]; [@bib52]). The consequences of activating PI3K signaling due to *PI3K* overexpression or loss of *PTEN* function has been extensively studied in *Drosophila* ([@bib33]; [@bib17]; [@bib25]; [@bib58]; [@bib13]; [@bib4]). Cells overexpressing *Dp110/PI3K* are enlarged and, in the fat body, increase their nutrient storage. This stockpiling of nutrients helps them to cell-autonomously bypass the nutritional requirements for cellular growth and DNA replication during amino acid deprivation ([@bib4]). In mitotic tissues, clones of *PTEN* mutant cells are enlarged, which is mainly caused by an increase in cell size ([@bib33]). However, given the importance of PTEN as a tumor suppressor, the overgrowth caused by the loss of PTEN is rather mild ([@bib17]; [@bib25]; [@bib13]).

Recently, it has been demonstrated that tumors lacking PTEN or with increased PI3K activity are resistant to dietary restriction ([@bib28]). This observation underscores the importance of understanding the intrinsic changes in early tumors caused by the microenvironment. Furthermore, it remains largely unknown how a growing tumor impacts on its environment.

In this study, we attempted to mimic early events in tumor development by inducing clones of *PTEN* mutant cells under conditions in which nutrients become limiting. We show that cells lacking PTEN switch from hypertrophic growth to hyperplastic growth under nutrient restriction (NR). This hyperproliferation occurs at the expense of neighboring wild-type cells, probably by competition for local and systemic pools of nutrients and other growth-promoting factors.

Results {#s2}
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Reduced insulin signaling is required for a proper starvation response {#s2-1}
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To assess the impact of starvation on survival, developmental timing, and weight, we reared larvae on food with varying yeast content. Yeast is the main source for micronutrients and amino acids in standard fly media. We observed a three-phase starvation response ([Figure 1A,B](#fig1){ref-type="fig"}). Our standard medium contains 100 g/l yeast. Down to 50 g/l yeast, larvae can be regarded as fully fed since the developmental time, weight, and survival were not affected. At a yeast concentration range between 50 g/l and 10 g/l, the development was gradually delayed and the weight decreased. This phase can be regarded as mild starvation, since survival was not compromised down to 25 g/l yeast concentration. Reduction of the yeast concentration to and below 10 g/l increased the developmental time from first instar to pupa and severely decreased body weight. Since mortality was also increased, this phase is regarded as severe starvation. Based on these results, the turning point between mild starvation and severe starvation is approximately at 10 g/l concentration of yeast.10.7554/eLife.00380.003Figure 1.Response to yeast starvation in wild-type and insulin signaling defective *Drosophila*.(**A** and **B**) Control flies reared on standard culture medium with varying yeast concentrations. (**A**) Weight reduction in males (blue line) and females (red line). (**B**) Time from first instar to pupariation (black line) and the survival rates of first instar larvae to pupariation (green line). (**C**) Adult dry weight of wild-type, *PKB* and *PTEN* mutants reared on 100 g/l and 10 g/l yeast food, respectively. Weight difference (in %) with respect to control (*y w*) on 100 g/l yeast food is depicted in each column. (**D**) Size comparison of female control flies *(y w)* and *PKB* hypomorphic mutants reared on 100 g/l and 10 g/l food, respectively. (**E**) Larvae of *y w* and a *PTEN* hypomorphic combination (*PTEN*^*117*^*/PTEN*^*100*^) reared on 100 g/l (late L3) and 10 g/l (mid L3). (**F**) Eye and wing discs of the larvae from (**E**).**DOI:** [http://dx.doi.org/10.7554/eLife.00380.003](10.7554/eLife.00380.003)10.7554/eLife.00380.004Figure 1---source data 1.Adult dry weight; survival L1 to pupariation; pupariation time; weight analysis of IIS mutants.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.004](10.7554/eLife.00380.004)

We next subjected hypomorphic *PKB* and *PTEN* mutants to 100 g/l and 10 g/l yeast food, respectively ([Figure 1C--F](#fig1){ref-type="fig"}). Interestingly, fully fed hypomorphic *PKB* mutants weighed approximately the same as wild-type flies reared on 10 g/l yeast food. When reared on 10 g/l yeast food, *PKB* mutants were further delayed but only mildly decreased in size and weight ([Figure 1C,D](#fig1){ref-type="fig"}). In contrast, hypomorphic *PTEN* larvae and adult flies were larger than wild-type controls under normal food conditions ([Figure 1E](#fig1){ref-type="fig"}). The prospective adult tissues, the eye and wing imaginal discs, were larger compared to control discs under both conditions ([Figure 1F](#fig1){ref-type="fig"}). *PTEN* hypomorphic larvae were highly sensitive to a reduction in yeast content, and although still larger, they did not survive to pupariation when reared on 10 g/l yeast food ([Figure 1C](#fig1){ref-type="fig"}). This is consistent with the findings that larvae with randomly induced PI3K overexpression clones are starvation sensitive ([@bib4]). Thus, under NR conditions, insulin signaling needs to be dampened to allow for survival of the organism.

Imaginal cells lacking PTEN gain a strong proliferative advantage under starvation conditions {#s2-2}
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In contrast to the situation at the organismal level, tumors with elevated PI3K activity are starvation resistant ([@bib28]). To mimic the clonal nature of cancer, we analyzed the loss of *PTEN* function in clones generated in mitotic tissues (imaginal discs) by hsFlp/FRT-mediated mitotic recombination ([@bib19]; [@bib60]). This system enables the generation of single genetically marked epithelial cells mutant for a defined tumor suppressor. Subsequent mitoses give rise to a patch of cells ('clone') devoid of the tumor suppressor. The clones can be tracked throughout development due to genetically encoded markers (usually GFP). In a typical experiment, we induced clones by a heat shock 24 hr after egg laying. Subsequently, the larvae were split into two populations and transferred to vials with yeast concentrations of 100 g/l and 10 g/l, respectively. The discs were then dissected to analyze the clones either at the same time point or at the same developmental stage ([Figure 2A](#fig2){ref-type="fig"}).10.7554/eLife.00380.005Figure 2.*PTEN* mutant cells are resistant to starvation and have a growth advantage upon NR.(**A**) Schematic drawing of the clonal induction with hsFlp/FRT-mediated mitotic recombination. After egg laying, larvae were allowed to grow for 24 hr on yeast paste. After the heat shock, larvae were distributed on different food conditions (e.g., 100 g/l and 10 g/l yeast). The time points of dissection are depicted in green. (**B**) Third instar eye discs bearing hsFlp/FRT *PTEN* mutant and control clones (marked by the absence of GFP) of larvae reared under varying yeast concentrations. (**C**) Eye discs with hsFlp/FRT *PTEN* mutant and control clones (marked by the absence of GFP) induced at the same time and conditions, reared on 10 g/l yeast food, and dissected at the indicated age of the clones. (**D**) Eyes bearing eyFlp/FRT *PTEN* mutant and control clones (marked by the absence of pigmentation) of animals reared on 100 g/l and 10 g/l yeast food, respectively. (**D′**) Quantification of the respective eye sizes. (**E**) Scanning electron micrographs of eyes almost exclusively composed of *PTEN* mutant or control tissue of animals reared on 100 g/l and 10 g/l food, respectively, and the quantification of ommatidia size (**E′**) and number (**E′′**) from *PTEN* mutant and control eyes.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.005](10.7554/eLife.00380.005)10.7554/eLife.00380.006Figure 2---source data 1.Eye size; eye measurements of SEM pictures.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.006](10.7554/eLife.00380.006)10.7554/eLife.00380.007Figure 2---figure supplement 1.Severe starvation induces malformations in adult eyes bearing *PTEN* clones.The structure of adult eyes carrying *PTEN* clones from animals raised on 10 g/l yeast food is unaltered. By contrast, severe distortions are observed in animals raised on 5 g/l yeast food. Magnifications of distorted parts of the eye are indicated with red boxes. The clones are marked by the absence of pigmentation.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.007](10.7554/eLife.00380.007)10.7554/eLife.00380.008Figure 2---figure supplement 2.Severe starvation affects architecture of discs bearing *PTEN* clones.Discs bearing *PTEN* clones (marked by the absence of GFP) have a lobed appearance in larvae on 10 g/l yeast food, and they are severely distorted with polyp-like outgrowths of hollow appearance (white arrow) in larvae on 5 g/l yeast food. On the right: close-ups of polyp-like structures indicated with white arrows.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.008](10.7554/eLife.00380.008)10.7554/eLife.00380.009Figure 2---figure supplement 3.Severely overgrown *PTEN* clones tend to collapse.Melanized scars (indicated with white arrows) are sometimes observed in adult structures, probably as remnants of collapsed *PTEN* clones in animals reared on 5 g/l yeast food.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.009](10.7554/eLife.00380.009)10.7554/eLife.00380.010Figure 2---figure supplement 4.*PTEN* mutant overgrowth is cell autonomous and does not affect differentiation.Eye sections of the adult eyes bearing control and *PTEN* clones from animals reared on 100 g/l and 10 g/l yeast food, respectively. The clones are marked by the absence of pigmentation.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.010](10.7554/eLife.00380.010)10.7554/eLife.00380.011Figure 2---figure supplement 5.*PTEN* clones have a growth advantage in wing discs under starvation.The starvation-dependent overgrowth of *PTEN* clones (marked by the absence of GFP) is not restricted to the eye imaginal disc but is also observed in wing imaginal discs.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.011](10.7554/eLife.00380.011)10.7554/eLife.00380.012Figure 2---figure supplement 6.*PTEN* mutant cells rapidly respond to the yeast content in the food.Food switch experiments reveal that a short starvation period early after clone induction is sufficient to result in the overgrowth phenotype of *PTEN* mutant tissue under starvation. The time points of the shifts are indicated; clones are marked by the absence of pigmentation.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.012](10.7554/eLife.00380.012)

We monitored the growth behavior of clones of *PTEN* mutant cells (henceforward called *PTEN* clones) in larvae reared under decreasing yeast concentration in the culture medium. As previously published, *PTEN* clones were enlarged in fed larvae but they did not severely impact on the structure of the imaginal discs and of the adult eyes ([@bib17]; [@bib13]) ([Figure 2B](#fig2){ref-type="fig"} and [Figure 2---figure supplement 1](#fig2s1){ref-type="fig"}). Under mild starvation conditions (30 g/l yeast), *PTEN* clones were further increased in size, resulting in an overall increase in disc size as compared to discs harboring control clones. Reducing the yeast concentration to 20 g/l and below led to a massive size increase of the *PTEN* clones and to a concomitant reduction of the surrounding tissue. Furthermore, the *PTEN* clones had fused and rendered the discs lobed in appearance ([Figure 2B](#fig2){ref-type="fig"} and [Figure 2---figure supplement 2](#fig2s2){ref-type="fig"}). At 5 g/l yeast concentration, the eye discs with *PTEN* mutant tissue were severely overgrown with polyp-like structures protruding from the discs ([Figure 2---figure supplement 2](#fig2s2){ref-type="fig"}). The resulting adult eyes displayed strong malformations and outgrowths, indicating a loss of epithelial integrity ([Figure 2---figure supplement 1](#fig2s1){ref-type="fig"}, red squares). In some cases, no clones could be recovered in the adult eyes, probably due to a collapse of the *PTEN* clones resulting in melanized scars ([Figure 2---figure supplement 3](#fig2s3){ref-type="fig"}, white arrow). Tangential eye sections did not reveal any structural defects in ommatidial arrangement ([Figure 2---figure supplement 4](#fig2s4){ref-type="fig"}). *PTEN* mutant cells overgrow in a cell-autonomous manner as the enlarged ommatidia were exclusively composed of mutant cells. The overgrowth phenotype of *PTEN* clones is not specific to the eye; it was also observed in other imaginal tissues like the wing disc ([Figure 2---figure supplement 5](#fig2s5){ref-type="fig"}).

Since starvation causes a developmental delay, the overgrowth of *PTEN* clones could be a consequence of the prolonged growth period. We therefore analyzed the *PTEN* clones at earlier time points (36 hr, 48 hr, and 60 hr after induction; [Figure 2C](#fig2){ref-type="fig"}). Already 36 hr after clone induction, the *PTEN* clones were increased in size, and the overgrowth of *PTEN* clones and the reduction in wild-type tissue were fully apparent after 60 hr ([Figure 2C](#fig2){ref-type="fig"}), excluding the developmental delay as a main cause of the overgrowth.

To investigate whether the early stages of starvation are crucial for the *PTEN* clones to overgrow, we performed 'food switch' experiments by transferring larvae with *PTEN* clones from starvation (10 g/l) to normal (100 g/l) food and vice versa at given time points. We used the eyeless-(ey)Flp/FRT system to induce clones specifically in the eye imaginal tissues early during larval development. When the transfer from starvation to normal food occurred within the first 72 hr, the overgrowth of the *PTEN* clones was efficiently rescued. Later shifts were no longer effective in suppressing the overgrowth. Consistently, it was sufficient to transfer larvae within the first 48 hr from normal to starvation food to produce the overgrowth phenotype. After this time point, *PTEN* clones did not acquire the full growth advantage over the surrounding tissue, confirming the importance of the initial stages for *PTEN* clones to develop the overgrowth phenotype ([Figure 2---figure supplement 6](#fig2s6){ref-type="fig"}).

We quantified the *PTEN* induced overgrowth by measuring the eye size of eyFlp/FRT-induced *PTEN* clones ([Figure 2D,D′](#fig2){ref-type="fig"}). Similar to the hsFlp/FRT-induced clones, the eyFlp/FRT-induced *PTEN* clones and the entire eyes were slightly enlarged in flies raised on normal food. Under starvation conditions, the mutant tissue occupied most of the adult eye and the wild-type tissue was severely reduced. This overrepresentation of the mutant tissue resulted in an absolute increase of eye size under starvation as compared to normal food conditions ([Figure 2D,D′](#fig2){ref-type="fig"}).

To investigate the effects of reduced nutrition on ommatidia size and number, we generated eyes almost completely composed of *PTEN* mutant tissue by means of the eyFlp/FRT cell lethal system and analyzed them by scanning electron microscopy ([Figure 2E--E′′](#fig2){ref-type="fig"}). Under normal food conditions, the eye size increase was mainly caused by larger ommatidia (+27%), and to a minor extent by more ommatidia (+14%). Under 10 g/l yeast food, the ommatidial size was roughly proportionally reduced in wild-type and *PTEN* mutant eyes. Intriguingly, whereas the ommatidia number was decreased by 6% in wild-type eyes, it was massively increased in *PTEN* mutant eyes (+44%). Since the composition of the *PTEN* mutant ommatidia remained unchanged, the size and number of ommatidia reflects cell size and cell number. Thus, the *PTEN* mutant tissue displays a switch from hypertrophy to hyperplasia.

The growth advantage of *PTEN* mutant cells is not due to cell competition {#s2-3}
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Cell competition, a process that results in the elimination of suboptimal cells from a growing tissue ([@bib35]; [@bib49]; [@bib50]), could contribute to the overgrowth of *PTEN* clones. 'Winner' cells actively eliminate 'loser' cells by inducing apoptosis and thereby take over the tissue ([@bib38]; [@bib37]; [@bib11]). If *PTEN* mutant cells acted as supercompetitors, the surrounding tissue would suffer from apoptosis. We therefore monitored apoptosis in eye imaginal discs bearing *PTEN* clones by cleaved Caspase-3 and TUNEL staining ([Figure 3A,B](#fig3){ref-type="fig"}). Whereas only few apoptotic cells were detected in the heterozygous tissue under both fed and starving conditions, increased levels of apoptosis were observed within the overgrowing *PTEN* clones ([Figure 3A,A′](#fig3){ref-type="fig"}).10.7554/eLife.00380.013Figure 3.*PTEN* mutant cells are susceptible to cell death.(**A**) Cleaved Caspase-3 antibody staining (in red) of hsFlp/FRT *PTEN* clones (marked by the absence of GFP) in eye imaginal discs of larvae reared under the indicated conditions. The yellow dashed line indicates the morphogenetic furrow. About one third of the *PTEN* clones contain apoptotic cells. (**A′**) Quantification of the apoptotic tissue area (positive for cleaved Caspase-3 immunostaining) in the *PTEN* mutant and the surrounding tissue (*PTEN*^*117*^/+, +/+), respectively, relative to the total area of the respective cell populations. (**B**) TUNEL staining (in red) on eye imaginal discs harboring 48 hr control and *PTEN* clones reveals high apoptotic levels within the *PTEN* clones but only residual apoptosis in surrounding tissue or control discs. (**C**) Expression of anti-apoptotic *p35* within *PTEN* clones (marked by GFP) enhances the overgrowth potential of *PTEN* mutant tissue. (**D**) Inhibition of apoptosis in the dorsal half of the eye discs by expression of *p35* under the control of *DE-Gal4* (marked by RFP, red) with randomly induced *PTEN* clones (marked by the absence of GFP) does not rescue the surrounding (GFP positive) tissue. (**E**) Inhibition of JNK-mediated apoptosis in the sister clones of the *PTEN* clones does not rescue the surrounding tissue. The different clones are marked as depicted with the colors in the labeling (green: GFP positive, white: GFP negative).**DOI:** [http://dx.doi.org/10.7554/eLife.00380.013](10.7554/eLife.00380.013)10.7554/eLife.00380.014Figure 3---source data 1.Tissue size; apoptotic area; ratio apoptotic area/tissue size.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.014](10.7554/eLife.00380.014)10.7554/eLife.00380.015Figure 3---figure supplement 1.Blocking cell death in *PTEN* clones enhances the overgrowth.Cleaved Caspase-3 antibody staining (in red) of *PTEN* and control clones expressing anti-apoptopic *p35* (positively marked by GFP) in eye imaginal discs of larvae reared under the indicated conditions. Cleaved Caspase-3 signal is exclusively observed in the *PTEN* mutant clones and efficiently suppressed by expression of *p35*.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.015](10.7554/eLife.00380.015)10.7554/eLife.00380.016Figure 3---figure supplement 2.The massive overgrowth of *PTEN* clones under harsh starvation conditions correlates with high levels of apoptosis.Outgrowing structures observed on 5 g/l yeast food are exclusively composed of *PTEN* mutant tissue (marked by the absence of GFP) and show high levels of cell death.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.016](10.7554/eLife.00380.016)

Since the overgrowth is fully apparent at 60 hr after clone induction, we investigated *PTEN* clones at 48 hr to exclude the possibility that cell competition eliminates the surrounding tissue at this early stage ([Figure 3B](#fig3){ref-type="fig"}). Again, most of the apoptosis was observed in the mutant tissue, arguing against a major role of cell competition in the overgrowth phenotype. Expression of *p35* or of dominant negative JNK (*bsk*^*DN*^; [@bib1]) exclusively in the *PTEN* clones (by means of the MARCM system) efficiently blocked apoptosis and enhanced the overgrowth ([Figure 3C](#fig3){ref-type="fig"}, [Figure 3---figure supplement 1](#fig3s1){ref-type="fig"}, and not shown).

To further address the role of apoptosis in the surrounding tissue, we expressed *p35* in the dorsal half of the eye and randomly induced *PTEN* clones throughout the disc ([Figure 3D](#fig3){ref-type="fig"}). Consistent with the above results and the apoptosis pattern, *PTEN* clones overgrew even more in the dorsal compartment, and the surrounding tissue still got strongly reduced. Thus, the cells neighboring *PTEN* clones are not eliminated by apoptosis.

We also tested for the requirement of JNK signaling in the tissue neighboring the *PTEN* clones. hsFlp/FRT clones of *bsk^1^* over a chromosome carrying a GFP marker and a *PTEN* mutation were induced. In this way, mitotic recombination events generate two adjacent twin spots mutant for *bsk^1^* and *PTEN,* respectively. If *PTEN* cells attained the growth advantage by inducing JNK-mediated cell death in the twin spot, the overgrowth would be suppressed and the twin spot should grow larger. However, *PTEN* clones were overgrown irrespective of blocking JNK signaling in neighboring cells ([Figure 3E](#fig3){ref-type="fig"}).

Under severe starvation (5 g/l yeast content), the *PTEN* mutant polyp-like structures outgrowing from the eye discs exhibited very high levels of apoptosis ([Figure 3---figure supplement 2](#fig3s2){ref-type="fig"}). In the corresponding adult eyes, we often observed scars that were probably left behind by the collapsing clones ([Figure 2---figure supplement 3](#fig2s3){ref-type="fig"}). Those eyes contained more wild-type cells, indicating that the lost *PTEN* mutant tissue was compensated for. Thus, *PTEN* mutant cells are running at the edge of survival under limited nutrient conditions. They start to disobey the organ boundaries but are prone to apoptosis.

*PTEN* mutant cells have elevated PIP3 levels, highly active PKB, and require TORC1 activity to overgrow {#s2-4}
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To gain a better understanding of the downstream events contributing to the overgrowth of *PTEN* clones, we monitored the levels of the second messenger PIP3 in discs containing *PTEN* clones by means of the tGPH reporter, a GFP--PH domain fusion protein with high affinity for PIP3 ([@bib4]) ([Figure 4A](#fig4){ref-type="fig"}). The GFP--PH domain fusion protein was strongly localized at the membrane under both fed and NR conditions in *PTEN* clones. In contrast, the reporter signal was diffuse in the cytoplasm in the surrounding tissue under both conditions. Removal of *Insulin receptor* (*InR*) function from the *PTEN* mutant tissue only slightly reduced the overgrowth. Thus, in the absence of PTEN, the cellular PIP3 levels are sufficient to sustain the overgrowth regardless of the upstream signaling ([Figure 4---figure supplement 1](#fig4s1){ref-type="fig"}). 10.7554/eLife.00380.017Figure 4.Interaction of insulin and TOR signaling with *PTEN* clones under starvation.(**A**) tGPH reporter (green) reveals high levels of PIP3 in *PTEN* clones (clones marked by absence of LacZ in red) under food conditions indicated. (**B**) P-PKB (red) is strongly increased in *PTEN* clones (marked by the absence of GFP) under normal conditions as well as under starvation. (**C**) *PTEN* overgrowth under normal and starvation conditions is strictly dependent on PKB activity. (**D**) Overexpression of *FoxO* results in a small eye under starvation conditions. This phenotype is suppressed by co-knockdown of *PTEN*. (**D′**) Quantification of eyes from (**D**). (**E**) Cherry-tagged FoxO localizes to the cytoplasm under normal food conditions and moves to the nucleus under starvation. This nuclear shuttling is prevented in *PTEN* clones. Clones are positively marked by Cherry (in red). (**F**) Inhibiting TORC1 activity by overexpression of *Tsc1/2* in *PTEN* clones (positively marked by GFP) suppresses the overgrowth. The suppression is evident already at 72 hr after clone induction. (**G**) Adult eyes showing the suppression of the overgrowth associated with *PTEN* clones by overexpression of *Tsc1/2* under starvation under standard conditions and under starvation (**G′**).**DOI:** [http://dx.doi.org/10.7554/eLife.00380.017](10.7554/eLife.00380.017)10.7554/eLife.00380.018Figure 4---source data 1.Eye size; shoulder size; eye/shoulder ratio; ommatidia number.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.018](10.7554/eLife.00380.018)10.7554/eLife.00380.019Figure 4---figure supplement 1.Genetic interactions between *PTEN* and IIS/TOR signaling components.Comparison of adult eyes bearing clones mutant for *InR*, *FoxO*, *Rheb*, *PI3K92E*, *PTEN* or combinations of *PTEN* with *InR*/*FoxO/Rheb/PI3K92E* under 100 g/l and 10 g/l yeast food conditions. Removal of *FoxO* slightly suppresses the overgrowth phenotype of *PTEN* mutant tissue under starvation. Similarly, a partial suppression is observed in *PTEN InR* and *PTEN PI3K92E* double mutants. Complete suppression is observed in *PTEN Rheb* double mutants.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.019](10.7554/eLife.00380.019)10.7554/eLife.00380.020Figure 4---figure supplement 2.Insulin and TOR signaling is modulated in *PTEN* clones in response to starvation.Western blots on eye imaginal disc tissue. On the left: *PTEN* mutant discs have strongly elevated P-PKB levels. Upon starvation, P-PKB levels are reduced but still much higher than in the control under normal conditions. Surprisingly, PKB levels are strongly reduced in the *PTEN* mutant tissue under starvation. On the right: *PTEN* mutant discs display high levels of P-S6K that strongly drop in starved animals. β-Tubulin served as loading control.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.020](10.7554/eLife.00380.020)10.7554/eLife.00380.021Figure 4---figure supplement 3.The effects of *PTEN* knockdown and *FoxO* overexpression neutralize each other.*GMR-Gal4* mediated expression of *PTEN*-*RNAi*, *FoxO* or both under 100 g/l yeast food conditions and quantification of the eye size normalized to shoulder size.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.021](10.7554/eLife.00380.021)10.7554/eLife.00380.022Figure 4---figure supplement 4.Overexpression of *FoxO* does not suppress *PTEN* mutant overgrowth.Overexpression of *FoxO* in *PTEN* clones (MARCM system, positively marked by GFP) does not suppress the *PTEN* mutant tissue overgrowth under both under normal and starvation conditions.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.022](10.7554/eLife.00380.022)10.7554/eLife.00380.023Figure 4---figure supplement 5.*PTEN* clones require TORC1 activity to overgrow.Size measurements of eyes bearing *PTEN* and control clones co-expressing *Tsc1/2* of animals reared on 100 g/l and 10 g/l yeast food.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.023](10.7554/eLife.00380.023)10.7554/eLife.00380.024Figure 4---figure supplement 6.Reducing TORC1 activity suppresses *PTEN* mutant overgrowth.Overexpression of *Tsc1*/2 in *PTEN* clones (positively marked by GFP) suppresses the overgrowth phenotype without reducing the P-PKB levels (red).**DOI:** [http://dx.doi.org/10.7554/eLife.00380.024](10.7554/eLife.00380.024)10.7554/eLife.00380.025Figure 4---figure supplement 7.*PTEN* mutant cells require TORC1 activity to acquire a proliferative advantage under starvation.Scanning electron micrographs of eyes generated with the eyFlp/FRT cell lethal system and almost exclusively composed of *PTEN*, *TOR*, *PTEN TOR* mutant or control tissue of animals reared on 100 g/l and 10 g/l yeast food, respectively. On the right side: Quantification of eye size, ommatidia number and ommatidia size. Note that the *PTEN TOR* double mutant eyes are very convex. As the projection of the eye onto the plane of the picture is measured, the indicated eye size is an underestimate.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.025](10.7554/eLife.00380.025)

Similarly, elevated levels of phosphorylated PKB (P-PKB) were found at the membrane in *PTEN* clones at 100 g/l and 10 g/l yeast content ([Figure 4B](#fig4){ref-type="fig"}). These results were further confirmed by a Western blot analysis comparing discs exclusively composed of *PTEN* mutant tissue with discs bearing wild-type clones under normal food conditions ([Figure 4---figure supplement 2](#fig4s2){ref-type="fig"}). Due to the extremely small size of the wild-type discs under NR, we were not able to generate enough tissue for the analysis. Although the total levels of PKB were decreased in *PTEN* mutant tissue under both conditions as compared to the control under normal food conditions, the P-PKB levels were elevated. Thus, high PIP3 levels result in robust PKB activation under standard and starvation conditions. Consistently, reducing or removing *PKB* function by using a PH-domain mutant ([@bib54]) and a kinase-dead PKB ([@bib53]), respectively, completely suppressed the *PTEN* overgrowth under normal as well as under starvation conditions ([Figure 4C](#fig4){ref-type="fig"}).

The transcription factor FoxO is an important target of PKB. Upon high PI3K signaling activity, FoxO is phosphorylated by PKB and sequestered in the cytoplasm, and FoxO-mediated transcription of growth-suppressing genes is thus inhibited ([@bib7]; [@bib30]; [@bib8]). FoxO was localized in the cytoplasm in *PTEN* mutant tissue under both conditions, whereas starvation induced the shuttling of FoxO into nuclei in the control tissue ([Figure 4E](#fig4){ref-type="fig"}). It has been shown that overexpression of *FoxO* decreases eye size, which is exacerbated under starvation conditions ([@bib27]; [@bib31]). Consistent with the inactivation of FoxO in *PTEN* mutant cells, the FoxO-induced eye size reduction was not observed in *PTEN* clones ([Figure 4D](#fig4){ref-type="fig"} and [Figure 4---figure supplements 3 and 4](#fig4s3 fig4s4){ref-type="fig"}).

Signaling via Target of Rapamycin complex 1 (TORC1) promotes growth in response to nutrients. High TORC1 activity boosts cellular growth, at least in part via the phosphorylation of S6K and 4E-BP (reviewed in [@bib22]). *PTEN* mutant cells did respond to starvation by reducing the levels of S6K and of phosphorylated S6K (P-S6K), as revealed by Western blot analysis on discs bearing eyFlp/FRT cell lethal clones of *PTEN* ([Figure 4---figure supplement 2](#fig4s2){ref-type="fig"}). PKB signaling activity was also slightly reduced as compared to standard conditions ([Figure 4B](#fig4){ref-type="fig"} and [Figure 4---figure supplement 2](#fig4s2){ref-type="fig"}). We next inhibited TORC1 activity selectively in *PTEN* clones by co-overexpression of Tuberous Sclerosis Complex-1 and -2 (Tsc1/2)---which together form a complex with GTPase activating protein (GAP) activity towards the small GTPase Rheb, an essential activator of TORC1 ([@bib14]; [@bib62])---or by removing *Rheb* function. Reducing TORC1 activity suppressed the overgrowth of the *PTEN* mutant tissue ([Figure 4F,G, 4G′](#fig4){ref-type="fig"} and [Figure 4---figure supplement 5](#fig4s5){ref-type="fig"}). P-PKB levels, however, were increased in the mutant tissue under both fed and NR conditions ([Figure 4---figure supplement 6](#fig4s6){ref-type="fig"}), which is in agreement with the negative feedback regulation of TORC1 on the activation of PKB ([@bib43]; [@bib29]). Eyes mutant for *PTEN* and a hypomorphic allele of *TOR* were reduced to the size of control eyes at both fed and starved conditions, indicating that the overgrowth of *PTEN* clones strictly depends on normal TORC1 function ([Figure 4---figure supplement 7](#fig4s7){ref-type="fig"}). Interestingly, whereas the size reduction of *PTEN* clones caused by impaired TORC1 function was primarily due to smaller ommatidia under normal conditions, ommatidia size was only slightly affected under starvation. By contrast, the hyperproliferation observed in *PTEN* clones under NR was almost completely abolished ([Figure 4---figure supplement 7](#fig4s7){ref-type="fig"}). Thus, TORC1 is indispensable for the switch to hyperproliferation observed in *PTEN* mutant tissue upon NR.

Knockdown of the amino acid transporter Slimfast suppresses the overgrowth of *PTEN* mutant cells {#s2-5}
-------------------------------------------------------------------------------------------------

The cationic amino acid transporter Slimfast (Slif) has been shown to activate TOR signaling in the fat body, and it appears to be an important component of a systemic nutrient sensor mechanism ([@bib10]). Slif function is also required in mitotic tissues, as clones in which *Slif* has been knocked down (*Slif*^*anti*^) are reduced in size, possibly due to reduced TORC1 activation. Since TORC1 is required for the overgrowth of *PTEN* clones, knocking down *Slif* should suppress this phenotype in a similar way as observed for *Tsc1/2* overexpression. Whereas expressing *Slif*^*anti*^ in *PTEN* clones resulted in a slight reduction of the mutant tissue under normal conditions, it caused a nearly complete elimination of the *PTEN* mutant tissue under starvation conditions, while not affecting the control clones ([Figure 5A](#fig5){ref-type="fig"}). The loss of *PTEN* mutant tissue was accompanied by a massive increase in apoptosis, and GFP-positive (and thus *PTEN* mutant) cellular remnants were scattered throughout the disc ([Figure 5B](#fig5){ref-type="fig"}), suggesting that *PTEN* clones collapsed after an initial overgrowth. Indeed, early overgrowth of *PTEN* clones and high P-PKB signals were observed under starvation ([Figure 5C](#fig5){ref-type="fig"} upper panel). Later, *Slif* function became limiting in *PTEN* clones that were subsequently eliminated ([Figure 5A,C](#fig5){ref-type="fig"} lower panel). Intriguingly, the structure of the resulting adult eyes that had lost the *PTEN* clones was completely normal, indicating that the surrounding tissue was able to compensate for the loss of the clones ([Figure 5D,D′](#fig5){ref-type="fig"}). The effects that the reduction of Slimfast exerted on *PTEN* clones are distinct from those resulting from inhibiting TORC1 function, as overexpression of *Tsc1/2* suppressed the overgrowth of *PTEN* clones already at an early stage ([Figure 4F](#fig4){ref-type="fig"}). This may indicate that, rather than the activation of TORC1, another aspect of Slif function---probably the amino acid influx itself---is critical for the survival of the *PTEN* mutant tissue.10.7554/eLife.00380.026Figure 5.*PTEN* mutant tissue critically depends on the amino acid transporter Slimfast.(**A**) *PTEN* clones co-expressing *Slif*^*anti*^ collapse and disappear under starvation, whereas *Slif* reduction does not affect control clones. (**B**) TUNEL staining reveals high apoptosis levels (in red) in cells with *Slif*^*anti*^ expression. Reducing the levels of Slif eliminates *PTEN* mutant cells by apoptosis. (**C**) Dying 110 hr-old *PTEN* clones expressing *Slif*^*anti*^ lose the P-PKB signal (red), although they initially overproliferate (72 hr-old clones) indistinguishably from *PTEN* mutant tissue. (**D** and **D′**) Adult eyes with unmarked control, *PTEN* and *PTEN* plus *Slif*^*anti*^ clones under standard (**D**) and starvation conditions (**D′**). The reduction of Slif levels rescues the overgrowth associated with *PTEN* mutant eyes, especially under starvation conditions. All clones in the discs are positively marked by GFP.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.026](10.7554/eLife.00380.026)10.7554/eLife.00380.027Figure 5---figure supplement 1.Autophagy does not contribute to the initial survival of *PTEN* clones with reduced Slif levels under starvation.Inhibiting autophagy (by means of *Atg5-RNAi*) in *PTEN* clones with reduced Slif function (positively marked by GFP) neither impacts on their initial overgrowth nor on their later collapsing under starvation. Blocking apoptosis (by means of *p35* expression) in *PTEN* clones with reduced Slif (positively marked by GFP) prevents the *PTEN* mutant cells from being eliminated.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.027](10.7554/eLife.00380.027)10.7554/eLife.00380.028Figure 5---figure supplement 2.Inhibition of cell death in *PTEN* clones with reduced Slif prevents the initial collapsing in eye discs.Higher magnification pictures of the undead *PTEN* mutant cells. Some large (and thus *PTEN* mutant) cells have lost GFP expression (white arrows).**DOI:** [http://dx.doi.org/10.7554/eLife.00380.028](10.7554/eLife.00380.028)10.7554/eLife.00380.029Figure 5---figure supplement 3.Blocking autophagy and apoptosis in *PTEN* clones with reduced Slif does not prevent them from dying.Adult eye phenotypes of the clones shown in figure supplement 1. *PTEN* clones with reduced Slif function are not recovered in adult eyes upon inhibition of autophagy or apoptosis under starvation conditions. White arrows indicate malformations reminiscent of the collapsed clones.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.029](10.7554/eLife.00380.029)

We also assessed whether the initial overgrowth of the *PTEN* clones with reduced Slif function could be attributed to autophagy that maintains the levels of amino acids and thereby sustains TORC1 activity. However, reducing Atg5, a key component of the autophagic machinery, did not impact on the initial growth of *PTEN* clones with reduced Slif function ([Figure 5---figure supplements 1 and 3](#fig5s1 fig5s3){ref-type="fig"}). Blocking apoptosis (by means of *p35* expression) in *PTEN* clones with diminished Slif function delays the collapse of the clones as the clones can still be detected in imaginal discs ([Figure 5---figure supplements 1 and 2](#fig5s1 fig5s2){ref-type="fig"}) but are absent from adult eyes ([Figure 5---figure supplement 3](#fig5s3){ref-type="fig"}).

*PTEN* clones affect the neighboring tissue and the organism in a non-autonomous way {#s2-6}
------------------------------------------------------------------------------------

We also observed non-autonomous effects of the *PTEN* clones on the surrounding tissue. P-PKB levels in the wild-type tissue neighboring with *PTEN* clones were reduced as compared to the tissue neighboring with control clones ([Figure 6---figure supplement 1](#fig6s1){ref-type="fig"}). This reduction is more apparent under normal conditions, which can be attributed to the already low P-PKB levels in discs of starved animals.

We were wondering whether this cell non-autonomous phenomenon only affects closely neighboring tissue or whether it acts at a longer range. Knocking down *PTEN* in the dorsal part of the eye not only resulted in its size increase, but also reduced the size of the ventral part as compared to control eyes ([Figure 6A,A′](#fig6){ref-type="fig"}). This growth-reducing effect within the eye was already visible under standard conditions but further enhanced on 10 g/l yeast food. In contrast to the null mutant situation, RNAi-mediated knockdown of *PTEN* did not result in enhanced overgrowth under starvation as compared to standard conditions, probably due to residual PTEN in the knockdown situation.10.7554/eLife.00380.030Figure 6.Systemic effects of *PTEN* clones on peripheral tissues.(**A**) *PTEN* was knocked down in the dorsal part of the eye by means of *DE-Gal4*. (**A′**) Quantification of the sizes of the ventral and dorsal halves. Percentage indicated in the bars represents the size reduction with respect to the respective part of control eyes under normal conditions. (**B**) Flies with *PTEN* mutant heads have smaller bodies as judged by shoulder area (s.a.) under starvation conditions. (**C**) Wings of flies with *PTEN* mutant heads under starvation are smaller than wings of control flies. (**C′**) Quantification of the wings from (**C**). (**D**) Size of fat body nuclei of larvae with *PTEN* mutant eye discs is decreased under starvation as compared to the control. (**D′**) Quantification of nuclear size from (**D**). *PTEN* mutant heads were generated by the eyFlp/FRT cell lethal system. Statistical analyses were done with Student's *t*-test (two tailed).**DOI:** [http://dx.doi.org/10.7554/eLife.00380.030](10.7554/eLife.00380.030)10.7554/eLife.00380.031Figure 6---Source data 1.Dorsal eye size; shoulder size; wing size; nuclear size in fat bodies.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.031](10.7554/eLife.00380.031)10.7554/eLife.00380.032Figure 6---figure supplement 1.*PTEN* mutant tissue influences PKB signaling in the neighboring tissue.The tissue surrounding *PTEN* clones (marked by the absence of GFP) displays a reduction in P-PKB levels (red) as compared to the tissue surrounding wild-type clones. Note that the nuclei of the cells adjacent to *PTEN* clones under starvation are more densely packed, indicative of a reduced cell size.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.032](10.7554/eLife.00380.032)

The analysis of flies with heads mostly composed of *PTEN* mutant tissue revealed similar non-autonomous effects on the sizes of other organs and of the entire body. A decrease in shoulder area (as a measure for body size) was observed under starvation but not under normal conditions ([Figure 6B](#fig6){ref-type="fig"}). Consistently, the wing size and the fat body nuclear size were reduced, especially in the animals that were raised under starvation ([Figure 6C,C′,D,D′](#fig6){ref-type="fig"}). Thus, the growth-reducing non-autonomous effect of *PTEN* mutant tissue acts systemically.

The observed non-autonomous effects suggest that the *PTEN* mutant tissue efficiently competes with other larval tissues for common resources to support their massive growth, thereby launching a vicious cycle in the neighboring tissue that gets further starved and reduced. If our interpretation was correct, *PTEN* clones should overgrow less under starvation when growth signaling is maintained in neighboring cells. We attempted to promote IIS by expressing the ligand *Dilp-2* in eye imaginal discs ([@bib5]; [@bib26]). Whereas expressing *Dilp-2* under *ey-Gal4* control did not affect control eyes, it suppressed the overgrowth caused by the loss of *PTEN* under starvation conditions. Furthermore, it restored the growth of both the tissue surrounding the *PTEN* clones and peripheral tissue ([Figure 7A](#fig7){ref-type="fig"}).10.7554/eLife.00380.033Figure 7.A reduction in growth signaling in the direct neighborhood and in peripheral tissues is required for the overgrowth of *PTEN* mutant cells.(**A**) Restoring insulin signaling by means of *Dilp-2* expression (driven by *ey-Gal4*) suppresses the overgrowth of *PTEN* mutant cells under starvation. The eyes get smaller and the growth of the surrounding and the peripheral tissue is restored. The expression of *Dilp-2* has no effect on control eyes. Quantification of eye size (**A′**) and shoulder area (**A′′**) from (**A**). (**B**) Systemic reduction of growth by ubiquitous expression of *Imp-L2* with *arm-Gal4* under standard conditions decreases the size of the control eyes but enhances *PTEN* mutant overgrowth. (**B′**) Quantification of eye size and shoulder area from (**B**).**DOI:** [http://dx.doi.org/10.7554/eLife.00380.033](10.7554/eLife.00380.033)10.7554/eLife.00380.034Figure 7---Source data 1.Eye size, shoulder size; ommatidia number; ommatidia size.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.034](10.7554/eLife.00380.034)10.7554/eLife.00380.035Figure 7---figure supplement 1.Growth properties of *PTEN* mutant tissue are influenced by the neighboring tissue.*PTEN* clones (marked by the absence of GFP) overgrow less under starvation when neighboring with *Tsc1* clones (marked by the absence of LacZ, pink), indicating that both clonal populations compete for common resources.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.035](10.7554/eLife.00380.035)10.7554/eLife.00380.036Figure 7---figure supplement 2.Autonomous and non-autonomous effects of reducing TORC1 activity on the overgrowth of *PTEN* clones.Reducing TORC1 signaling (using the hypomorphic allele *TOR*^*EP2353*^) specifically in *PTEN* mutant cells suppresses their proliferative advantage and restores growth of the surrounding normal tissue on both 100 g/l and 10 g/l yeast food. Conversely, when neighboring with sister clones mutant for *TOR*, *PTEN* clones outcompete the surrounding tissue already at standard conditions and overgrow more under starvation. The different clones are marked as depicted with colors in the labeling (orange: containing red/orange pigmentation, black: lacking red/orange pigmentation). Below: Quantification of eye sizes.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.036](10.7554/eLife.00380.036)10.7554/eLife.00380.037Figure 7---figure supplement 3.*PTEN* mutant cells neighboring with *TOR* mutant twin clones acquire a proliferative advantage early in larval development.Eye discs with eyFlp/FRT *PTEN* and *PTEN* over *TOR* mutant clones (*PTEN* cells are positively marked by GFP), dissected at the indicated time points after egg deposition.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.037](10.7554/eLife.00380.037)10.7554/eLife.00380.038Figure 7---figure supplement 4.Wild-type cells neighboring with *TOR* or *PKB* mutant cells gain a growth advantage.Eye discs with hsFlp/FRT *TOR* and *PKB* clones (marked by the absence of GFP). Note that the wild-type twin clones (bright green due to two copies of *ubiGFP*) are strongly overgrown.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.038](10.7554/eLife.00380.038)10.7554/eLife.00380.039Figure 7---figure supplement 5.*PTEN* mutant cells sense the growth reduction in their neighborhood and proliferate faster under starvation.Scanning electron micrographs of eyes generated with the eyFlp/FRT ubiGFP system bearing *TOR*, *PTEN*, *PTEN* over *TOR* mutant or control clones of animals reared on 10 g/l yeast food. When neighboring with sister clones mutant for *TOR*, *PTEN* cells overgrow more under starvation exclusively due to an increase in cell number. On the rights side: Quantification of eye size, ommatidia number and ommatidia size.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.039](10.7554/eLife.00380.039)10.7554/eLife.00380.040Figure 7---figure supplement 6.Systemic reduction of growth signaling induces a starvation-like response in *PTEN* mutant cells.Systemic reduction of IIS (achieved by *arm-Gal4 UAS-Imp-L2*) enhances the overgrowth of *PTEN* clones that are neighboring with *TOR* mutant sister clones. The bigger eyes are composed of more but smaller ommatidia. Below: Quantification of eye size, ommatidia number and ommatidia size. Note that the increase in cell number in *PTEN* clones neighboring with *TOR* mutant cells is an underestimate because the reference eyes contain many very small (*TOR* mutant) ommatidia.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.040](10.7554/eLife.00380.040)

We also analyzed the consequences of introducing cells with deregulated TORC1 activity---and thus enhanced cellular growth--in the neighborhood of *PTEN* clones. To this end, we simultaneously generated *PTEN* clones and clones of *Tsc1* mutant cells ([Figure 7---figure supplement 1](#fig7s1){ref-type="fig"}). Interestingly, the mutant clones did not have additive effects on overgrowth. The introduction of *Tsc1* clones into the neighborhood of *PTEN* clones rather reduced their overgrowth under starvation. The discs were completely composed of either *Tsc1* or *PTEN* mutant tissue, as the clones did not heavily overlap. Thus, when neighboring with a tissue that also has a growth advantage, *PTEN* clones themselves experience competition for common resources.

To investigate whether the overgrowth of *PTEN* clones is enabled by a non-autonomous influence from the starved neighboring tissue, we genetically starved the surrounding tissue of *PTEN* clones by reducing TORC1 activity. We induced eyFlp/FRT clones of *PTEN* with sister clones mutant for *TOR* ([Figure 7B](#fig7){ref-type="fig"}, [Figure 7---figure supplements 2 and 3](#fig7s2 fig7s3){ref-type="fig"})*.* The resulting eyes were completely composed of *PTEN* mutant tissue already under standard conditions. However, the eyes were smaller as compared to eyes bearing *PTEN* clones only.

We also observed that clones of *TOR* mutant cells generated in otherwise wild-type eyes were massively underrepresented. The adult eyes were almost completely composed of wild-type twin clone tissue ([Figure 7B](#fig7){ref-type="fig"}), suggesting that not only *PTEN* mutant cells with a high growth potential but also wild-type cells neighboring with cells impaired in TORC1 function attain a growth advantage and proliferate in place of the slower growing cells. Similar observations were made with mutations impinging on IIS activity (e.g., *PKB*; [Figure 7---figure supplement 4](#fig7s4){ref-type="fig"}). Thus, relative differences in IIS activity cause a differential growth behavior.

We next tested how the combination of genetically starved neighboring cells (mutant for *TOR*) with real starvation during development impacts on the growth behavior of *PTEN* clones. Interestingly, when in competition with *TOR* mutant cells, *PTEN* clones did overgrow under NR as opposed to normal conditions ([Figure 7---figure supplement 2](#fig7s2){ref-type="fig"}). The overgrowth was even more pronounced than without *TOR* mutant neighbors, and it was apparent already early during larval development ([Figure 7---figure supplement 3](#fig7s3){ref-type="fig"}). SEM analysis revealed that the additional overgrowth was primarily caused by more cells ([Figure 7---figure supplement 5](#fig7s5){ref-type="fig"}).

Finally, we wondered whether systemically dampening IIS would allow the overgrowth of *PTEN* clones under normal conditions. We ubiquitously expressed *Imp-L2*, which encodes a secreted antagonist of Dilp-2 ([@bib24]). In this context, *PTEN* clones did overgrow, irrespective of whether they were neighboring with *TOR* mutant cells or wild-type cells ([Figure 7B](#fig7){ref-type="fig"}). The overgrowth of the *PTEN* clones caused a further reduction in body size, as evidenced by decreased shoulder width ([Figure 7B](#fig7){ref-type="fig"}). Cells devoid of *PTEN* also reacted to the decrease in IIS activity (by slightly reducing cell size) but the massive increase in cell number caused the total overgrowth ([Figure 7---figure supplement 6](#fig7s6){ref-type="fig"}). Thus, systemically decreasing IIS enhances the proliferative potential of *PTEN* mutant cells.

Discussion {#s3}
==========

Tumors with high PI3K pathway activity are associated with increased resistance to starvation. Here, we describe how the loss of the tumor suppressor PTEN contributes to early clonal expansion. *PTEN* mutant cells tolerate and survive starvation in a clonal situation in *Drosophila* imaginal discs. This response is completely dependent on high PKB and sustained TORC1 activities within the *PTEN* clones. *PTEN* mutant cells also acquire a growth advantage under starvation conditions at the expense of wild-type cells in the immediate neighborhood and in the entire organism.

It has previously been demonstrated that activation of PI3K (or of TORC1) results in starvation insensitivity in endoreplicative tissues (ERTs) ([@bib4]). The role of activated PI3K in starvation resistance of *Drosophila* epithelia is also not unprecedented. Anaplastic Lymphoma Kinase (ALK)-dependent activation of PI3K is necessary for bypassing the amino acid requirement in growing neuroblasts (neural progenitors) under NR conditions (brain sparing) ([@bib9]). However, imaginal tissues are not spared upon starvation, arguing against an important function of ALK in mediating growth of imaginal discs under NR conditions.

Mitotically active imaginal disc cells with high PIP3 levels respond differently to starvation as compared to corresponding cells in the ERTs. *PTEN* mutant imaginal disc cells do react to starvation by reducing their size like control cells do. The cell size reduction is proportional: *PTEN* mutant cells are still enlarged with respect to control cells under starvation. However, the cell size reduction is compensated for by a massive increase in proliferation, causing a net increase in mutant tissue. In fact, the *PTEN* mutant tissue is absolutely enlarged under starvation conditions.

*PTEN* mutant cells therefore gain a growth advantage and can take over a complete organ when the surrounding tissue is starved. This advantage is neither due to a prolonged growth period (*PTEN* clones are increased in size early after clone induction) nor to a complete insensitivity to starvation. It rather reflects a change in their mode to respond to a limited access to nutrients: *PTEN* mutant cells switch from hypertrophic to hyperplastic growth under starvation. This tolerance towards increased proliferation could favor the selection for secondary mutations that enhance proliferation and thus tumor progression.

To our surprise, *PTEN* clones display increased levels of apoptosis, and selective inhibition of apoptosis within the clones results in tremendous hyperplastic overgrowth. The induction of apoptosis in *PTEN* mutant tissue contrasts the known pro-survival function of PKB ([@bib47]). However, it indicates that the starved *PTEN* mutant cells, despite their proliferative advantage, exist on the edge of survival and are highly susceptible to apoptosis. Our findings also suggest that acquisition of factors preventing apoptosis would strongly enhance the growth of tumors lacking PTEN.

How can *PTEN* mutant cells grow at the expense of the non-mutant tissue? Comparing the P-PKB levels in the heterozygous and wild-type tissue revealed a reduction in the tissue surrounding *PTEN* clones as compared to tissue adjacent to control clones. Furthermore, cells neighboring with *PTEN* mutant tissue are smaller than those in the discs containing control clones. Thus, *PTEN* clones impact on IIS activity in the surrounding tissue, thereby reducing its growth potential. This appears to be sufficient to explain the strong reduction of the surrounding tissue, since the process of ousting the neighboring tissue is completed as early as 60 hr after clonal induction, when the disc still contains relatively few cells. This non-autonomous growth reduction is not restricted to the local neighbors in a cell--cell contact-dependent manner, but rather affects all tissues of the organism.

The impact of *PTEN* mutant tissue on its surroundings suggests that the cells compete for common factors, most likely growth factors and nutrients. Under starvation, the circulating levels of growth factors and nutrients are strongly reduced, and thereby can become limiting ([@bib9]). *Drosophila* insulin-like peptides (DILPs) produced by the insulin-producing cells (IPCs) in the brain stimulate the growth of imaginal disc cells via the InR. Starvation reduces the secretion of DILPs from the IPCs, and thus reduces growth ([@bib15]). Removing *InR* function in *PTEN* clones only mildly affects the overgrowth. Thus, despite lacking the signal input via InR, these cells are able to accumulate PIP3, probably because of the basal activity of PI3K. Therefore, low levels of circulating DILPs are sufficient to boost PIP3 levels and PKB activity in *PTEN* mutant cells under starvation.

The cationic amino acid transporter Slimfast (Slif) has been described as an upstream activator of TORC1 ([@bib10]). We show that, in contrast to inhibiting TORC1 activity, reducing Slif does not block the initial overgrowth of *PTEN* clones under starvation. However, it causes the overgrown clones to collapse. We speculate that the high metabolic demands of *PTEN* mutant cells require an efficient amino acid uptake, which is blocked by reducing Slif. Thus, *PTEN* mutant cells rely on their ability to efficiently compete for nutrients and growth factors under starvation conditions with their direct neighbors and the peripheral tissue. Since the reduction of Slif affects the growth of the wild-type and the *PTEN* mutant tissue in a differential manner, this amino acid transporter could represent a target for a dosage-dependent drug therapy of tumors with PI3K activation.

Our results suggest the following sequence of events when a single cell embedded in a mitotic tissue loses the tumor suppressor PTEN ([Figure 8](#fig8){ref-type="fig"}). The loss of *PTEN* function triggers high PIP3 levels, thereby activating PKB and TORC1, thus stimulating cell growth and division. Under starvation conditions, circulating growth factors and nutrients are scarce. In response to these limited resources, both wild-type and *PTEN* mutant cells adjust their growth with respect to cellular size. In sharp contrast to wild-type clones, *PTEN* clones retain high PI3K activity and strongly increase their cell number. This hyperplastic clonal overgrowth depends on withdrawing nutrients from the neighborhood and ultimately the entire organism. The active scavenging of resources is dependent on efficient amino acid transporters (Slif), which also ensure that TORC1 remains active. During their entire growth, cells are at the brink of death as evidenced by the high apoptosis rate within the *PTEN* clones. Thus, enhanced PIP3 levels in cells that lost the tumor suppressor PTEN not only leads to starvation resistance at the cellular level, but also suppresses growth in their direct tissue neighborhood and in peripheral tissues by competing for nutrients and growth factors. Our findings demonstrate how limiting nutrient conditions enhance the proliferative potential of *PTEN* mutant cells, independent of additional genetic alterations.10.7554/eLife.00380.041Figure 8.Model of hyperplastic overgrowth of *PTEN* mutant tissue under starvation.(**A**) Under standard conditions (normal food), the *PTEN* mutant tissue overgrows in a hypertrophic manner. The tissue is enlarged because of larger *PTEN* mutant cells. (**B**) Under starvation conditions, the *PTEN* mutant tissue is metabolically more active and outcompetes the surrounding wild-type tissue, resulting in a hyperplastic overgrowth. The size of the tissue is further increased because of more *PTEN* mutant cells. The *PTEN* mutant tissue is susceptible to apoptosis, and it depends on the function of the amino acid transporter Slimfast. Under both conditions, the *PTEN* mutant tissue exhibits high insulin signaling activity and is dependent on the functions of PKB and TORC1.**DOI:** [http://dx.doi.org/10.7554/eLife.00380.041](10.7554/eLife.00380.041)

Materials and methods {#s4}
=====================

Fly media and stock keeping {#s4-1}
---------------------------

1 liter *Drosophila* medium contains 100 g of fresh yeast, 55 g cornmeal, 10 g wheat flour, 75 g sugar and 8 g bacto agar, here referred to as standard medium (or 100 g/l yeast food). Starvation food (or 10 g/l yeast food) was generated by reducing the amount of yeast without altering the other ingredients. All crosses and experiments were performed at 25°C under non-crowding conditions.

Mutants and transgenes {#s4-2}
----------------------

To generate a viable hypomorphic situation for *PTEN*, the null allele *PTEN*^*117*^ and the hypomorphic allele *PTEN*^*100A*^ were used in heteroallelic combinations ([@bib42]). For clonal analysis, *FRT40 PTEN*^*117*^ and *FRT82 Tsc1*^*Q87X*^ ([@bib41]; [@bib56]) were used. To generate null mutant clones for *PTEN* on the third chromosome, the genomic rescue construct *FRT82 PTEN*^*genomic\ rescue*^ *ubiGFP* ([@bib13]) was used in a null mutant *PTEN*^*117*^ background. Knockdown of *PTEN* was achieved using the VDRC line 101475 in combination with *GMR-Gal4* and *DE-Gal4* ([@bib39]). For genetic interaction studies with the insulin and TOR pathways, the following alleles were used: *FRT82 PKB*^*1*^ and *FRT82 PKB*^*3*^ ([@bib54]), *FRT82 InR*^*5545*^ ([@bib12]), *FRT82 PI3K92E*^*2H1*^ ([@bib20]), *FRT82 FoxO*^*25*^ ([@bib27]), *FRT FoxO*^*Δ94*^ ([@bib51]), *FRT40 TOR*^*2L19*^ ([@bib41]), *FRT40 TOR*^*EP2353*^ ([@bib61]) and *FRT82 Rheb*^*2G5*^ ([@bib55]). The following transgenic fly lines were used: *GMR-Gal4 Thor*^*1*^*; EP-dFoxO* ([@bib27]), *UAS-cherry dFoxO*, *UAS-Tsc1/2* ([@bib56]), *UAS-Dilp2* ([@bib5]), *UAS-Imp-L2* ([@bib24]) and *UAS-Atg5*^*RNAi*^ ([@bib48]). For blocking apoptosis, *FRT40 bsk*^*1*^ ([@bib44]), *UAS-p35* ([@bib21]) and *UAS-bsk*^*DN*^ ([@bib1]) were used. To monitor PI3K activity, a tGPH reporter was used ([@bib4]). The amino acid transporter Slimfast was silenced using *Slif*^*anti*^ ([@bib10]). A full list of alleles and transgenes used is provided in [Supplementary file 1](#SD7-data){ref-type="supplementary-material"}.

Stress experiments {#s4-3}
------------------

Flies were crossed in standard rearing vials for 3 days, transferred to laying cages, and allowed to lay eggs on apple agar plates (11 hr in most of the experiments, 3 hr for fat body analysis). For induction of eyFlp/FRT clones, eggs were distributed to the different food conditions immediately, whereas for induction of hsFlp/FRT clones, eggs were allowed to hatch, and they were heat-shocked before distribution. For quantification of survival, dead embryos were counted 24 hr after seeding to the food, and survival of the pupae was recorded.

Phenotypic analyses {#s4-4}
-------------------

All phenotypic analyses on adult flies were performed on females (unless indicated otherwise), and measurement of nuclear size in fat bodies was done on female larvae. Size and number of the ommatidia in SEM pictures, size of the eyes, shoulders, wings, and fat body nuclei were measured using Photoshop CS3. Student's *t*-test (two-tailed) was used to test for significance in all the quantification experiments.

For determination of dry weight, flies were dried at 95°C for 5 min and individually weighed with a Mettler Toledo MX5 microbalance.

Clonal analyses {#s4-5}
---------------

Mutant clones in eye and wing imaginal discs were generated with *y, w, hsFlp; FRT40* or *FRT82* flies. Clones were induced during the first instar (heat shock for 15 min at 37°C, 38 hr after egg deposition \[AED\]), and larvae were dissected in the third instar before wandering, unless otherwise indicated. For positively marked *PTEN* knockdown clones, *Actin Flp-out Gal4* ([@bib40]) and the MARCM ([@bib32]) system were used. Clones generated by the *Actin Flp-out Gal4* technique were induced during the first larval instar (heat shock for 10 min at 37°C, 38 hr AED), and larvae were dissected in the third instar before wandering. Eye-specific clones were generated using the eyFlp/FRT system. The exact genotypes are indicated in [Supplementary file 1](#SD7-data){ref-type="supplementary-material"}.

Immunohistochemistry, Western blotting and histology {#s4-6}
----------------------------------------------------

Larval imaginal discs were fixed in 4% PFA (30 min, RT), permeabilized in 0.3% PBT (15 min, RT), blocked in 2% NDS in 0.3% PBT (1 hr, RT), incubated with the primary antibodies overnight (4°C), washed three times in 0.3% PBT, and incubated with secondary antibodies (1 hr, RT). The nuclei were visualized with 1:2000 DAPI in 0.3% PBT (15 min, RT). Antibodies used in this study were: rabbit α*-Drosophila* phospho-Akt/PKB Ser505 (1:300; Cell Signaling), rabbit α-cleaved Caspase 3 (1:300; Cell Signaling), mouse α-β-Galactosidase (1:300; Promega), Cy3- and Cy5-coupled α-mouse or α-rabbit IgG (1:300; Amersham). All the dilutions were made in blocking solution. TUNEL staining was carried out according to the manufacturer's protocol (ApopTag Red In Situ Apoptosis Detection KitS7165; Millipore).

Larval fat bodies were fixed in 8% PFA (45 min, RT) and stained with 1:50 Alexa Fluor 488 phalloidin in 0.2% PBT (90 min, RT; Molecular Probes) and 1:500 DAPI in 0.2% PBT (5 min, RT).

Western blots on L3 eye imaginal discs were performed according to standard protocols. Antibodies were α*-Drosophila* phospho-PKB Ser 505 (1:1000; Cell Signaling), α*-*PKB (1:1000; Cell Signaling), α-phospho-S6K (1:1000; Cell Signaling), α-S6K (1:2000; our own antibody), α-Tubulin (1:10,000; Sigma), HRP-conjugated α-mouse and α-rabbit IgG (1:10,000; Amersham).

Histological sections of the adult fly eyes were performed as previously described ([@bib3]).

Image acquisition {#s4-7}
-----------------

For the confocal images, a Leica SPE confocal laser scanning microscope was used. A Jeol JSM-6360LV microscope was used for scanning electron microscope pictures. For color pictures of larvae and adults, a KEYENCE VHX1000 digital microscope was used. For the pictures of the wings and the histological sections of the eyes, a Zeiss Axiophot Microscope was used.

Statistical analyses {#s4-8}
--------------------

In all the quantifications, Student's *t*-test (two-tailed) was used to test for significance. In each experiment, a minimum of nine individuals was measured for each genotype. Significance is indicated in the Figures using the following symbols: \*p\<0.05, \*\*p\<0.01, \*\*\*p\<0.001, n.s., not significant. Error bars represent the standard deviation. All measurement data are provided in the Source Data Files accompanying the Figures including statistical analyses ([Figure 1---source data 1](#SD1-data){ref-type="supplementary-material"}, [Figure 2---source data 1](#SD2-data){ref-type="supplementary-material"}, [Figure 3---source data 1](#SD3-data){ref-type="supplementary-material"}, [Figure 4---source data 1](#SD4-data){ref-type="supplementary-material"}, [Figure 6---source data 1](#SD5-data){ref-type="supplementary-material"}, [Figure 7---source data 1](#SD6-data){ref-type="supplementary-material"}).

Funding Information
===================

This paper was supported by the following grants:

-   Swiss National Science Foundation 31003A_125208 to Hugo Stocker.

-   ETH Zürich to Ernst Hafen.

We thank P Bansal, M Adlesic, A Baer, J Lüdke, A Straessle, I Vuillez for technical support; K Basler, B Edgar, D Pan, P Leopold, N Tapon, the Bloomington and VDRC stock centers for fly stocks; K Köhler for critical comments on the manuscript; M Aguet, W Krek, P Leopold, J Szabad and present and former members of the Hafen laboratory for discussions.

Additional information
======================

The authors declare that no competing interests exist.

KN, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.

GS, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.

EH, Conception and design, Drafting or revising the article.

HS, Conception and design, Analysis and interpretation of data, Drafting or revising the article.

Additional files
================

10.7554/eLife.00380.042

###### Genotypes of experimental animals.

**DOI:** [http://dx.doi.org/10.7554/eLife.00380.042](10.7554/eLife.00380.042)

10.7554/eLife.00380.043

Decision letter

Sabatini

David M

Reviewing editor

Whitehead Institute/Massachusetts Institute of Technology

,

United States

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see [review process](http://elife.elifesciences.org/review-process)). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

Thank you for choosing to send your work entitled "Nutrient restriction enhances the oncogenic potential of cells lacking the tumor suppressor PTEN in a mitotic tissue" for consideration at *eLife*. Your article has been evaluated by a Senior Editor and 3 reviewers, one of whom is a member of our Board of Reviewing Editors.

The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.

Substantive concerns to be addressed during revision:

1\) The finding that *PTEN* mutant tissues decrease the growth of wild-type tissues is very intriguing, but this aspect of the work is underdeveloped and needs to be investigated at a mechanistic level. First, levels of DILPs and specific nutrients in hemolymph from larvae or adults with *PTEN* loss in the eye should be measured, if possible. Alternatively, for the DILPs, the levels in the median neurosecretory (mNSCs) brain cells that secrete them can be quantitated as a reduction in circulating DILPs -- this usually correlates with an increase in DILPs in the mNSCs.

2\) Second, it is important to test if a boost in the levels of secreted DILPs is sufficient to rescue the growth defect of wild-type cells in animals with *PTEN*-null clones. In a similar vein, does an increase in Slimfast in wild-type cells ameliorate their growth defects in the context of *PTEN* mutant clones?

3\) To expand the scope of the work, it is important to determine if *Tsc1* mutant clones behave similarly to those with reduced PTEN function. Given the results in [Figure 6---figure supplement 1](#fig6s1){ref-type="fig"}, it appears that the necessary strains are available. The results of this experiment would increase the impact of this story by determining whether sustained TORC1 signaling is both necessary and sufficient for the enhanced clonal overgrowth (combined hypertrophy and hyperplasia) under nutrient restriction.

10.7554/eLife.00380.044

Author response

*1) The finding that* PTEN *mutant tissues decrease the growth of wild-type tissues is very intriguing, but this aspect of the work is underdeveloped and needs to be investigated at a mechanistic level. First, levels of DILPs and specific nutrients in hemolymph from larvae or adults with* PTEN *loss in the eye should be measured, if possible. Alternatively, for the DILPs, the levels in the median neurosecretory (mNSCs) brain cells that secrete them can be quantitated as a reduction in circulating DILPs -- this usually correlates with an increase in DILPs in the mNSCs*.

The reviewers raise a key question regarding the unknown mechanism of the observed competition between *PTEN* mutant and normal cells. Indeed, we considered measuring DILP levels. However, as DILP-2, the most potent growth- promoting DILP, is retained in the mNSCs upon starvation, it would be difficult to determine increased retention by immunostaining. For a reliable mass-spectrometric quantification, the amount of tissue obtained from our experimental setup is limiting.

As our genetic experiments pointed to an important role of amino acids, we measured amino acid concentrations in the hemolymph of larvae bearing eye- specific *PTEN* clones and of control larvae by gas chromatography coupled with mass spectrometry (GC-MS). However, the results we obtained are rather complex. It has been reported that the concentrations of alanine, isoleucine, leucine, and valine decrease upon acute starvation (Anaplastic lymphoma kinase spares organ during nutrient restriction in *Drosophila,* [@bib9]). In our experiment, only two of them, isoleucine and valine, exhibited the same tendency, whereas the concentration of alanine, the most abundant amino acid, even increased upon starvation. Leucine levels were not considerably affected. The results of a representative experiment are shown below in Author response image 1. The discrepancies may be a consequence of the differences in the experimental setups. In our experiment, we subjected the larvae to chronic starvation instead of acute starvation. We checked the changes at the end of a long-term process, which may no longer reflect the situation at the beginning of the starvation. It is also conceivable that changes in amino acid concentrations occur rather locally and not in the systemic pool in the hemolymph.Author response image 1.Concentrations of amino acids in the hemolymph of larvae bearing *PTEN* clones and control larvae under normal and starvation conditions.

We also assessed the starvation response in the fat body, a central organ in nutrient sensing. We performed a gene expression analyses for *InR* and *4EBP-1,* two genes known to be transcriptionally upregulated upon starvation. This experiment is expected to be more sensitive with respect to systemic changes than the measurement of DILPs in the mNSCs by immunostaining. We dissected fat bodies of the tightly staged larvae (egg laying for 5 hours) with *PTEN* mutant and control eye discs early in the third instar (72 h under standard conditions and 120 h under starvation conditions) to avoid any changes in gene expression associated with the metamorphosis processes taking place in the fat body. mRNA levels in fat bodies were determined by qRT-PCR. The levels of *InR* and *4EBP-1* mRNAs were consistently upregulated upon starvation. However, we did not detect any differences between the larvae bearing *PTEN* clones and the control larvae (Author response image 2). Again, changes in gene expression may have been alleviated by adaptation mechanisms occurring during chronic starvation. It is also possible that key events triggering the overgrowth of *PTEN* mutant tissue take place at the onset of starvation.Author response image 2.Expression levels of the starvation-induced genes *InR* and *4EBP-1* in the fat body of control larvae and larvae bearing *PTEN* clones (see text for explanations).

As the quantifications of amino acid levels and of starvation-responsive genes did not yield conclusive results, we resorted to a genetic experiment to support our notion that the growth properties of *PTEN* mutant tissue influence the growth of the surrounding and peripheral tissues, and vice versa. We attempted to genetically starve the larvae in order to mimic the starvation response in the animals. To this end, we systemically dampened IIS by ubiquitously expressing Imp-L2, a secreted antagonist of Dilp2 (by means of *arm-Gal4).* In this context, *PTEN* mutant clones did overgrow more, which was accompanied by a further reduction in body size. Thus, *PTEN* mutant cells compete with the surrounding normal cells for pools of systemic growth factors, and a systemic reduction in IIS enhances the overgrowth of *PTEN* mutant tissue. Closer analysis of the overgrown eyes by SEM revealed that cells devoid of PTEN reacted to systemically decreased IIS by slightly reducing their size, but a massive increase in cell number caused the net overgrowth. Thus, systemically decreasing IIS enhances the proliferative potential of *PTEN* mutant cells. These data are shown in the new [Figure 7B](#fig7){ref-type="fig"} and the accompanying [Figure 7---figure supplement 6](#fig7s6){ref-type="fig"}.

*2) Second, it is important to test if a boost in the levels of secreted DILPs is sufficient to rescue the growth defect of wild-type cells in animals with* PTEN*-null clones. In a similar vein, does an increase in Slimfast in wild-type cells ameliorate their growth defects in the context of* PTEN *mutant clones*?

We performed both experiments suggested by the reviewers. Expression of *Dilp2* by means of *ey-Gal4* in the context of *PTEN* clones restored the growth of the surrounding and peripheral tissue and suppressed the *PTEN* overgrowth, while not affecting control eyes. These results are shown in [Figure 7A](#fig7){ref-type="fig"}. Similar results were obtained by the expression of wild-type *InR* (data not shown).

An analogous experiment was performed with *Slimfast.* Expression of *Slif* (by means of *ey-Gal4)* slightly increased the sizes of control and *PTEN* mutant eyes, but this effect did not reach statistical significance (Author response image 3). Thus, the increased expression of Slif did not restore the growth of the surrounding tissue. However, as we do not know whether increasing the levels of the amino acid transporter Slif enhances its total activity, the experiment is not conclusive and we did not include it in the manuscript.Author response image 3.Overexpression of *Slif* does not rescue the growth of the tissue neighboring with *PTEN* clones.

*3) To expand the scope of the work, it is important to determine if* Tsc1 *mutant clones behave similarly to those with reduced PTEN function. Given the results in* [*Figure 6---figure supplement 1*](#fig6s1){ref-type="fig"}*, it appears that the necessary strains are available. The results of this experiment would increase the impact of this story by determining whether sustained TORC1 signaling is both necessary and sufficient for the enhanced clonal overgrowth (combined hypertrophy and hyperplasia) under nutrient restriction*.

We agree that the analysis of *Tsc1* mutant clones is an important issue. Cells devoid of Tsc1 are overgrowing under starvation conditions, similar to *PTEN* mutant cells. However, a thorough analysis revealed significant differences between *PTEN* and *Tsc1* clones. As this story is rather complex, we did not want to include it in the present manuscript.

Acknowledgement: The authors thank Manuel Hörl and Nicola Zamboni (Institute of Molecular Systems Biology, ETH Zürich) for the GC-MS analyses shown in Author response image 1.

[^1]: These authors contributed equally to this work.

[^2]: Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain.
