Target of Rapamycin Complex 2 regulates cell growth via Myc in Drosophila

Target of rapamycin (TOR) is an evolutionarily conserved serine/threonine protein kinase that functions as a central regulator of cellular growth and metabolism by forming two distinct complexes: TOR complex 1 (TORC1) and TORC2. As well as TORC1, TORC2 plays a key role in regulation of cell growth. But little is known about how TORC2 regulates cell growth. The transcription factor Myc also plays a critical role in cell proliferation and growth. Here we report that TORC2 and Myc regulate cell growth via a common pathway. Expression of Myc fully rescued growth defects associated with lst8 and rictor mutations, both of which encode essential components of TORC2. Furthermore, loss of TORC2 disrupted the nuclear localization of Myc, and inhibited Myc-dependent transcription. Together, our results reveal a Myc-dependent pathway by which TORC2 regulates cell growth.

seen in dmyc, lst8, and rictor mutant animals (the latter two encode essential components of TORC2), suggesting that Myc is an essential link between TORC2 and cell growth 7,9,23 .
Using the Drosophila model system, we found that both cellular and organismal growth defects of dmyc mutant animals were not exacerbated by the loss of LST8. Ectopic expression of dMyc completely rescued the growth defects of both lst8 and rictor mutant animals, including reduced body weight and shrunken eyes and wings. Moreover, the nuclear localization of dMyc was disrupted in lst8 or rictor mutant cells. Furthermore, gene expression profiling revealed that a large set of growth related genes was dysregulated in dmyc, lst8, and rictor mutant animals. Our findings suggested that Myc functions downstream of TORC2 to regulate cell growth.
Scanning electron microscopy (SEM). SEM was performed as described 9 . Briefly, heads were dissected from newly enclosed male flies reared at 25 °C, dehydrated with a series of ethanol dilutions, and immersed in hexamethyldisilazane. Samples were coated with gold/palladium and mounted after the solvent was evaporated off. The samples were examined with a JEOL JSM-5800 microscope. The area of 20 ommatidia in high-magnification SEM pictures was measured with ImageJ, and the mean ommatidium size was calculated.
Phosphate affinity SDS-PAGE and western blot. Mobility shift of phosphorylated dMyc protein was detected by phosphate affinity SDS-PAGE using acrylamide-pendant phos-tag (Phos-tag AAL-107). Briefly, 50-100 μ M phos-tag acrylamide and 100-200 μ M MnCl 2 were added to normal 6% polyacrylamide gel. After electrophoresis, the gel was washed with transfer buffer containing 1 mM EDTA for 10 min with gentle agitation, and then with transfer buffer without EDTA for 10 min. Proteins were transferred to Immobilon-FL transfer membranes (Millipore). For western blotting, pupae were homogenized in SDS sample buffer with a pellet pestle (Kimble-Kontes), and the proteins were fractionated using SDS-PAGE. The proteins were transferred to Immobilon-FL transfer membranes in Tris-glycine buffer. The blots were probed with mouse anti-HA 1:500 (Cell Signaling), mouse anti-dMyc (1:50), and rabbit anti-α tubulin 1:15000 (Sigma), and subsequently with IRDye 800-labeled anti-mouse IgG and IRDye 680-labeled anti-rabbit IgG (Licor). Signals were detected using an Odyssey infrared imaging system.

RNA-seq analysis.
Reads were mapped to the Drosophila genome (BDGP5.25) using TopHat (v2.0.10) by allowing up to two mismatches 27 . Expression of genes in the RNA-seq data was measured by calculating reads per kilobase per million mapped reads (RPKM). P-values to detect differential expression were calculated by cufflinks (v2.0.2) 28 . The criteria for differential expressed genes in a mutant were defined as more than two-fold change in RPKM and less than 0.01 in P-values. Gene ontology analysis was performed with DAVID online (http://david.abcc.ncifcrf.gov/) 29 .
Using the hs-FLP/FRT system, we next generated clones of mutant cells in heterozygous fat body tissues. Mutant clones were marked by the absence of red fluorescent protein (RFP) and we assessed larval fat body cell size by staining the tissue with phalloidin to visualize cell boundaries (Fig. 1C). Similar to body weight, loss of LST8, which completely disrupts TORC2 activities, reduced cell size to 76.72% of surrounding control cells, whereas loss of dMyc alone or LST8 and dMyc reduce cell size to 69.37% and 69.33% of control values, respectively (Fig. 1D). To compare growth rates between lst8 1 , dm P0 , and lst8 1 dm P0 cells in greater detail we used another mosaic system, in which lst8 1 dm P0 double mutant clones were generated in a lst8 1 or dm P0 single-mutant genetic background (Fig. 1E). Cell size analysis revealed that lst8 1 dm P0 fat body cells were smaller (80.97%) than neighboring lst8 1 cells. However, this size reduction was not seen when lst8 1 dm P0 double mutant cells were generated in a dm P0 genetic background (Fig. 1E,F), indicating that loss of dMyc exacerbated growth defects associated with lst8 1 , but loss of LST8 did not affect dm P0 cells.
We next generated double mutant lst8 1 dm P0 clones of cells in lst8 1 or dm P0 developing wing discs. Cell size was examined using fluorescence-activated cell sorting (FACS) and forward scatter (FSC) analysis was used to measure cell volumes. This analysis confirmed that loss of dMyc further reduced the size of lst8 1 mutant cells, but the loss of LST8 did not affect dm P0 mutant cells (Fig. 1G,H, left panels). DNA profiles demonstrated that loss of TORC2 and dMyc did not alter cell cycle phasing (Fig. 1G,H, right panels), consistent with previous findings that reductions in cell size associated with the loss of LST8 and dMyc do not result from changes in cell proliferation. These data suggest that dMyc is a potential downstream effector of the TORC2 signaling pathway to mediated cell growth. However, given the essential role of dMyc in organismal growth and development, disrupting TORC2 signaling through the loss of LST8 will likely not abrogate dMyc function completely. This may explain why reductions in cell size were more remarkable in dm P0 , and lst8 1 dm P0 mutant cells than in lst8 1 cells.

Myc functions downstream of TORC2 to regulate cell growth.
To determine whether dMyc functions downstream of TORC2 signaling, rescuing experiments were performed in which engrailed-Gal4 (en-Gal4) was used to drive expression of UAS-dmyc in the posterior compartment of developing wings in lst8 1 animals. Expression of dMyc in lst8 1 animals rescued cell growth within the posterior compartment without affecting the anterior compartment ( Fig. 2A,B).
Considering that different tissues exhibit differences in growth regulation, we next examined the adult compound eye to determine the general relationship between TORC2 and dMyc. Similar results were obtained in the eye. Overexpression of dMyc using GMR-Gal4 significantly increased ommatidial size in lst8 1 mutant eyes. Similar size effects were seen when dMyc was overexpressed in the wild-type background (Fig. 2C,D). These data support the hypothesis that dMyc participates in TORC2-driven cell growth. However, as overexpression of dMyc by the Gal4/UAS system causes overgrowth of cells 30 (Fig. 2C,D), dMyc and TORC2 might also drive cell growth via parallel pathways.
To more precisely assess the interaction between loss of TORC2 and dMyc overexpression, we moderately overexpressed dMyc using the tubulin promoter (Tub-dmyc). This construct did not drive cell overgrowth 31 , as wing size and body weight were unaffected by the ubiquitous expression of dMyc. In contrast, growth phenotypes of dm P0 animals were strongly rescued by Tub-dmyc (Fig. 2E,F). In lst8 1 , rictor Δ1 , and lst8 1 dm P0 mutant animals, the reduced body weight and shrunken wings were completely rescued by the Tub-dmyc transgene. As a negative control, growth reduction caused by pink1 depletion was unaffected by dMyc overexpression, suggesting that dMyc only rescues growth defects caused by disruptions in TORC2 signaling. These findings strongly supported of our hypothesis that Myc functions as a downstream effector of TORC2 to affect TORC2-mediated cell growth.

Loss of Myc function upon disruption of TORC2.
As a transcription factor, Myc predominantly localizes to the nucleus. As TORC2 might affect cell growth by modulating dMyc pathway, we hypothesized that TORC2 may affect the subcellular localization of dMyc. Immunolabeling experiments were performed using larval fat body tissue to monitor changes in dMyc localization. Antibodies against dMyc revealed that endogenous dMyc localized exclusively to the nucleus in wild-type larval fat body cell, while dMyc staining was dramatically reduced in dm P0 cells (Fig. 3A,B). In fat body tissue lacking LST8 or Rictor, a large portion of dMyc was detected outside of the nucleus, failing to overlap with Dapi (Fig. 3C,D).
Gene ontology classification of the 265 overlapping genes among lst8 1 , dm P0 , and rictor Δ1 are shown in Fig. 4C. Major gene ontology categories included protein metabolic process (18.87%), proteolysis (16.98%), and macromolecule catabolic process (9.81%), which are highly related to cell growth. Together these account for 45.66% of the overlapping genes. We further analyzed genes in the category: "protein metabolic process". A heat map plot comparing the four genotypes clearly demonstrates the highly similar patterns of expression for genes in this category (Fig. 4D). Transcription levels of these genes were further confirmed with qPCR ( Supplementary Fig. S1). We next analyzed genes that overlapping in lst8 1 , lst8 1 dm P0 , and rictor Δ1 but not in dm P0 mutant tissues. Among these 218 genes, there are a large portion of cell death related genes, indicating that TORC2 has additional functions independent of dMyc in some cellular events such as cell death (Supplementary Fig. S2). Moreover, among 836 genes that are down-regulated in the lst8 1 mutant flies, 101 genes are still down-regulated in the lst8 1 dm P0 mutant flies  Heatmap presentation of gene expression for genes in the "protein metabolic process" category. Relative gene expression is encoded in graduated colors from red (upregulated compared with wild type) to blue (downregulated compared with wild type) according to fold change.
when comparing with the dm P0 flies (Supplementary Fig. S3). Similarly, among 685 up-regulated genes in the lst8 1 mutant tissues, 71 genes are still up-regulated in the lst8 1 dm P0 double mutant tissues when comparing with the dm P0 tissues (Supplementary Fig. S3). Nevertheless, the highly consistent expression patterns among the 4 mutant stains suggests that TORC2 regulates dMyc transcriptional activity by modulating its nuclear localization.

Discussion
Here we describe a crosstalk between TORC2 and Myc, two regulators of cell growth. The genetic and cell biology studies in Drosophila place TORC2 upstream of MYC in a pathway that regulates cell growth. These results suggest that TORC2 inhibitors may represent effective therapies for treating Myc-driven cancers.
TOR kinase is a highly conserved protein kinase and a central regulator of cell growth. TORC1 has been extensively characterized, but the recent identification of a second TOR complex (TORC2) has complicated the TOR-regulated cell growth pathways. TORC2 regulates growth and metabolism in both mammals and invertebrates 6,8,9,32 . Recent findings indicate that ribosomes physically interact with TORC2 and that this interaction is required for mTORC2 activation. This suggests a critical role for TORC2 in cell growth regulation 33 . However, little is known about the regulation of mTORC2 signaling, or the downstream effectors that implement TORC2-mediated cell growth 4 . Given evidence that TORC2 regulates AKT/FOXO, AKT/FOXO signaling has been considered the major factor acting downstream of TORC2 to control growth. Our findings that AKT-or FOXO does not affect TORC2-mediated cell growth strongly argue against the AKT/FOXO pathway acting downstream of TORC2 in this context 9 .
In the present study, we found that MYC is required for TORC2-regulated cell growth. Mutations in lst8, rictor, or dmyc had similar growth defects, and lst8 dmyc double mutants did not have more severe growth phenotypes than dmyc mutants. Mosaic analysis in multiple cell types showed that lst8 dmyc double mutant cells had similar growth rates and cell sizes as dmyc mutant cells within the same animals. Moreover, we established that Myc functions downstream of TORC2 in cell growth. Growth phenotypes associated with loss of TORC2 in the retina, wing, fat body, and the entire body were rescued by the overexpression of dMyc.
Myc protein controls metabolism, cell growth, and proliferation by regulating genes transcribed by RNA Polymerase II, and by stimulating transcription by RNA Polymerases I and III 15 . As Myc is a transcription factor, pathways that regulate the subcellular localization of Myc likely affect its ability to regulate growth and metabolism 34 . Here we found that the lack of TORC2 activity is associated the cytoplasmic accumulation of dMyc. Consequently, many Myc target genes were dysregulated in lst8 and rictor mutant tissues. TORC2-mediated nuclear localization of Myc may represent a novel mechanism by which Myc activity is regulated.
As master regulators of cell growth and metabolism, TORC1 and Myc exhibit coordinated patterns of activity 35 . TORC1 activity is required for cancer cell survival, and TORC1 inhibition has remarkable therapeutic efficacy in Myc-driven hematological cancers 36 . In flies, inhibition of TORC1 by molecular inhibitors, genetic manipulations, or starvation leads to the post-transcriptional downregulation of dMyc followed by the repression of dMyc target genes 16,25 . In mammals, it has been reported that TORC1 activity is required for efficient c-MYC translation in TSC2-null Elt3 rat leiomyoma cells 37 , but opposite results have been reported for colorectal cancer cells, in which TORC1 inhibition by rapamycin treatment or knockdown of Raptor results in phosphorylation and accumulation of Myc 38 . Moreover, in Drosophila intestinal stem cells, excessive TORC1-driven growth in TSC mutants blocked dMyc-induced cell division 39 . These results challenge the notion that TORC1 inhibitors can be used as therapeutic drugs in Myc-driven cancers.
In some cases, such as hyperactivation of AKT signaling, TORC2 is required for proliferation of tumor cells and subsequent tumor growth 33,40 . The selective requirement for mTORC2 in tumor development suggests that mTORC2 inhibitors may be of substantial clinical utility 40 . The PI3K/AKT signaling cascade is known to regulate metabolic processes via discrete effectors, such as the TSC (tuberous sclerosis) complex and FOXOs. TORC2 inactivates the FOXO branch without affecting the TSC/TORC1 branch 41,42 . It has been demonstrated that FOXO inhibits MYC function to decrease mitochondrial function and to reduce ROS production 43,44 . Moreover, a central role for TORC2 in cancer metabolic reprogramming has been proposed, wherein mTORC2 signaling increases cellular c-Myc levels by acetylating FOXO independent of AKT 45 . Here we found that TORC2 controlled cell growth by regulating dMyc via nuclear localization. This regulation of dMyc by TORC2 is likely not through TORC2-mediated inactivation of FOXO, because mutations in lst8 or rictor do not affect dMyc transcription or translation. Moreover, previous reports that TORC2 does not regulate cell growth via AKT/FOXO support the model that TORC2 regulates cell growth via Myc independent of FOXO 9 . This finding suggests that TORC2 inhibitors may represent an effective way of treating Myc-driven cancers.