text:
- •High dietary sugar enhances Ras/Src-mediated transformation in Drosophila
- •Ras/Src-activated tumors evade diet-mediated insulin resistance
- •Insulin resistance evasion is due to Wingless-mediated insulin receptor upregulation
- •Rational targeting of multiple pathways can reduce diet-enhanced tumors
The risk of specific cancers increases in patients with metabolic dysfunction, including obesity and diabetes. Here, we use Drosophila as a model to explore the effects of diet on tumor progression. Feeding Drosophila a diet high in carbohydrates was previously demonstrated to direct metabolic dysfunction, including hyperglycemia, hyperinsulinemia, and insulin resistance. We demonstrate that high dietary sugar also converts Ras/Src-transformed tissue from localized growths to aggressive tumors with emergent metastases. Whereas most tissues displayed insulin resistance, Ras/Src tumors retained insulin pathway sensitivity, increased the ability to import glucose, and resisted apoptosis. High dietary sugar increased canonical Wingless/Wnt pathway activity, which upregulated insulin receptor gene expression to promote insulin sensitivity. The result is a feed-forward circuit that amplified diet-mediated malignant phenotypes within Ras/Src-transformed tumors. By targeting multiple steps in this circuit with rationally applied drug combinations, we demonstrate the potential of combinatorial drug intervention to treat diet-enhanced malignant tumors.
Graphical Abstract
The prevalence of metabolism-related diseases, including type 2 diabetes and obesity, has been increasing worldwide. Metabolic disease impacts body homeostasis, leading to a constellation of symptoms, including cardiovascular disease, blindness, neuropathy, and nephropathy. Recently, the American Diabetes Association and American Cancer Society jointly published a consensus report emphasizing accumulating evidence that patients with diabetes also show elevated risks of specific cancer types (
Giovannucci et al., 2010). Epidemiological studies have provided strong evidence for the association between cancer and metabolic diseases, including diabetes and obesity (
- Giovannucci E.
- Harlan D.M.
- Archer M.C.
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- Gapstur S.M.
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- Pollak M.
- Regensteiner J.G.
- Yee D.
Diabetes and cancer: a consensus report.
CA Cancer J. Clin. 2010; 60: 207-221
Barone et al., 2008,
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Long-term all-cause mortality in cancer patients with preexisting diabetes mellitus: a systematic review and meta-analysis.
Calle et al., 2003,
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Overweight, obesity, and mortality from cancer in a prospectively studied cohort of U.S. adults.
N. Engl. J. Med. 2003; 348: 1625-1638
Coughlin et al., 2004,
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Diabetes mellitus as a predictor of cancer mortality in a large cohort of US adults.
Am. J. Epidemiol. 2004; 159: 1160-1167
Inoue et al., 2006); patients with metabolic dysfunction have both a higher incidence of specific tumor types and a higher overall cancer-related mortality. For example, obese patients with progesterone-receptor-negative breast cancer had a higher risk of lymph node metastasis, suggesting that metabolic dysfunction can promote tumor aggressiveness (
- Inoue M.
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Diabetes mellitus and the risk of cancer: results from a large-scale population-based cohort study in Japan.
Arch. Intern. Med. 2006; 166: 1871-1877
Maehle et al., 2004). The increase in metabolic diseases worldwide highlights metabolic dysfunction as an increasing issue in cancer progression. However, the mechanisms by which metabolic dysfunction contributes to cancer progression remain poorly understood.
- Maehle B.O.
- Tretli S.
- Thorsen T.
The associations of obesity, lymph node status and prognosis in breast cancer patients: dependence on estrogen and progesterone receptor status.
Obesity and type 2 diabetes are typically associated with chronic hyperinsulinemia; levels of insulin in the blood rise to compensate for insulin resistance. Increased circulating insulin levels are in turn a risk factor for the development of hepatocellular carcinoma and colorectal cancer (
Donadon et al., 2009,
- Donadon V.
- Balbi M.
- Zanette G.
Hyperinsulinemia and risk for hepatocellular carcinoma in patients with chronic liver diseases and Type 2 diabetes mellitus.
Expert Rev. Gastroenterol. Hepatol. 2009; 3: 465-467
Kaaks et al., 2000). Together with the well-documented mitogenic effects of insulin (
- Kaaks R.
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- Lukanova A.
- Biessy C.
- Dechaud H.
- Rinaldi S.
- Zeleniuch-Jacquotte A.
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Serum C-peptide, insulin-like growth factor (IGF)-I, IGF-binding proteins, and colorectal cancer risk in women.
J. Natl. Cancer Inst. 2000; 92: 1592-1600
Ish-Shalom et al., 1997), this evidence suggests a role for hyperinsulinemia as a promoter of enhanced tumorigenesis in obese and diabetic patients. However, the development of insulin resistance in metabolism-related diseases raises a key question: how do tumors overcome insulin resistance to take advantage of increasing insulin levels? Here, we utilize Drosophila to explore the effects of high dietary sugar on tumor progression in vivo.
- Ish-Shalom D.
- Christoffersen C.T.
- Vorwerk P.
- Sacerdoti-Sierra N.
- Shymko R.M.
- Naor D.
- De Meyts P.
Mitogenic properties of insulin and insulin analogues mediated by the insulin receptor.
Diabetologia. 1997; 40: S25-S31
Feeding Drosophila a diet supplemented to 1.0 M sucrose led to insulin resistance, hyperglycemia, increased insulin levels (hyperinsulinemia), and accumulation of fat, phenocopying important aspects of type 2 diabetes (
Musselman et al., 2011). We used this model to explore the effects of high dietary sugar on tumor progression in vivo. On a normal diet, Csk/Src-dependent tumors were eliminated from the epithelium either alone or when paired with an oncogenic Ras isoform. In contrast, elevated dietary sugar strongly synergized with Ras/Src to generate large tumors associated with aggressive metastasis-like behavior. Using this model system, we investigated whether increased insulin levels underlie induction of malignant tumors. Importantly, Ras/Src-activated tumors remained sensitive to insulin in dietary conditions that promoted insulin resistance in surrounding tissues. We demonstrate that increased insulin/PI3K signaling in turn prevents apoptosis and promotes canonical Wingless/Wnt mitogenic signaling in Ras/Src tumors. Our results provide functional data that, in the face of high dietary sugar, Ras/Src-activated cells escape insulin resistance to exhibit enhanced growth that is dependent on Wingless/Wnt mitogenic signaling.
- Musselman L.P.
- Fink J.L.
- Narzinski K.
- Ramachandran P.V.
- Hathiramani S.S.
- Cagan R.L.
- Baranski T.J.
A high-sugar diet produces obesity and insulin resistance in wild-type Drosophila.
Dis. Model. Mech. 2011; 4: 842-849
High Dietary Sucrose Enhanced Tumor Growth
Feeding Drosophila a standard diet supplemented to 1.0 M sucrose (high dietary sucrose or "HDS") led to metabolic defects reminiscent of specific aspects of type 2 diabetes, including insulin resistance, hyperglycemia, elevated Drosophila insulin-like peptide (DILP) levels, and accumulation of body fat (
Musselman et al., 2011). To explore the link between metabolic dysfunction and tumor progression, we compared Drosophila cancer models fed HDS to those fed a control (0.15 M sucrose) diet. Combined elevation of Ras and Src pathway activities is a common motif in multiple cancer types, including breast, colorectal, and pancreatic cancer (
- Musselman L.P.
- Fink J.L.
- Narzinski K.
- Ramachandran P.V.
- Hathiramani S.S.
- Cagan R.L.
- Baranski T.J.
A high-sugar diet produces obesity and insulin resistance in wild-type Drosophila.
Dis. Model. Mech. 2011; 4: 842-849
Ishizawar and Parsons, 2004,
- Ishizawar R.
- Parsons S.J.
c-Src and cooperating partners in human cancer.
Cancer Cell. 2004; 6: 209-214
Morton et al., 2010). Experimentally, oncogenic K-Ras plus Src isoforms cooperate to accelerate the onset of pancreatic ductal adenocarcinoma in mice models, and Ras plus Src act together to promote tumorigenesis in Drosophila (
- Morton J.P.
- Karim S.A.
- Graham K.
- Timpson P.
- Jamieson N.
- Athineos D.
- Doyle B.
- McKay C.
- Heung M.Y.
- Oien K.A.
- et al.
Dasatinib inhibits the development of metastases in a mouse model of pancreatic ductal adenocarcinoma.
Gastroenterology. 2010; 139: 292-303
Shields et al., 2011,
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- Lau S.K.
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- Huang M.
- Schmedt C.
- Tarin D.
- et al.
Oncogenic Ras/Src cooperativity in pancreatic neoplasia.
Oncogene. 2011; 30: 2123-2134
Vidal et al., 2007). We further developed this model by pairing transgenes that express oncogenic ras1G12V in the eye with the genotypically null cskQ156Stop allele of Csk, a primary negative regulator of Src kinase. Clones were labeled with GFP to visualize tumor progression. In control eyes, GFP expression was comparable in control diet versus HDS, indicating that high-sucrose feeding did not alter transgene expression levels (Figures 1A and 1B ).
- Vidal M.
- Warner S.
- Read R.
- Cagan R.L.
Differing Src signaling levels have distinct outcomes in Drosophila.
Cancer Res. 2007; 67: 10278-10285
Genotypically csk null clones alone were rarely obtained when fed either a control diet or HDS (Figures 1C and 1D) due to Src-mediated apoptosis (
Vidal et al., 2006). Also, as previously reported (
- Vidal M.
- Larson D.E.
- Cagan R.L.
Csk-deficient boundary cells are eliminated from normal Drosophila epithelia by exclusion, migration, and apoptosis.
Dev. Cell. 2006; 10: 33-44
Pagliarini and Xu, 2003), targeting ras1G12V to the eye resulted in isolated benign tumors that in turn led to lethality as pupae (Figure 1E). ras1G12V animals fed HDS exhibited at most a mild increase in growth within the eye field (Figure 1F). Eyes with elevated Ras plus Src activity (ras1G12V;csk−/−) raised on a control diet displayed fewer and smaller GFP-positive clones compared to ras1G12V-expressing clones alone (Figures 1E and 1G), indicating that ras1G12V expression cannot overcome elimination of csk mutant cells.
- Pagliarini R.A.
- Xu T.
A genetic screen in Drosophila for metastatic behavior.
Science. 2003; 302: 1227-1231
By striking contrast, clones displayed strongly increased proliferation in 96.5% of ras1G12V;csk−/− larvae fed HDS. The result was striking overgrowth and a significantly enlarged GFP-positive eye field that dominated the head region (Figures 1H and 1I). This overgrowth had consequences on animal viability; a majority (77.5%) of ras1G12V;csk−/− animals fed HDS failed to initiate pupariation, instead dying as larvae with multiple secondary tumors (Figure S1A available online). Together, these results indicate that the metabolic status of the animals, as influenced by diet, strongly affects the outcome of Ras/Src-mediated tumorigenesis in the eye epithelium.
HDS feeding led to an ∼4 day delay in completion of larval development (
Musselman et al., 2011) (Figure S1B); therefore, phenotypes were compared between developmental-stage-matched animals fed HDS versus a control diet (late third-instar larvae). To rule out the possibility that enhanced tumor growth in HDS-fed animals was due to their older chronological age, we also analyzed progressive clone sizes in chronological-age-matched animals (Figures 1K–1S). At day 9 after egg laying (AEL), larval eye tissue was significantly smaller in HDS-fed animals than in age-matched controls (12.9%; Figures 1M′, 1N′, and S1C), but the percentage area occupied by GFP-positive clones was already significantly larger (13.5% in control diet and 42.8% in HDS; Figures 1M′, 1N′, and 1S). This difference widened over time; control clones decreased from 35.2% (day 7 AEL) to 13.5% (day 9 AEL) of total area while HDS clones progressively increased, reaching 58.9% at day 11 AEL and 93.7% at day 13 AEL (Figures 1K–1S). These analyses further support a role for HDS in enhancing growth of ras1G12V;csk−/− tumors.
- Musselman L.P.
- Fink J.L.
- Narzinski K.
- Ramachandran P.V.
- Hathiramani S.S.
- Cagan R.L.
- Baranski T.J.
A high-sugar diet produces obesity and insulin resistance in wild-type Drosophila.
Dis. Model. Mech. 2011; 4: 842-849
HDS, but Not High Fat, Induced Cancer Phenotypes
Diets containing 2.0 M glucose enhanced ras1G12V;csk−/− growth at a level similar to diets containing 1.0 M sucrose (Figures 1H and S2A). Other diets, including pure banana, that have similar carbohydrate content yielded similar tumor enhancement (Figure S2B), further emphasizing the specific role of high dietary carbohydrates. By contrast, ras1G12V;csk−/− tumors failed to show enhanced tumor growth in a calorie-matched high-fat diet (Figure S2C), indicating that high levels of dietary carbohydrates, but not high total caloric levels, are responsible for the enhanced cancer phenotype. This is consistent with observations that a high-carbohydrate diet in general leads to metabolic defects in Drosophila not observed with calorie-matched high-fat or high-protein diets (
Musselman et al., 2011).
- Musselman L.P.
- Fink J.L.
- Narzinski K.
- Ramachandran P.V.
- Hathiramani S.S.
- Cagan R.L.
- Baranski T.J.
A high-sugar diet produces obesity and insulin resistance in wild-type Drosophila.
Dis. Model. Mech. 2011; 4: 842-849
HDS Promoted Aspects of Invasive Migration and Distant Secondary Tumors
Basement membrane degradation by matrix metalloproteases (MMPs) is a key early step in the initiation of cell spreading and metastasis (
Deryugina and Quigley, 2006). Eye epithelia from ras1G12V;csk−/− animals fed a control diet displayed an intact basement membrane as assessed by Laminin A (Figure 2A). HDS feeding led to broad loss of Laminin A; loss was already evident at day 9 AEL (Figure 2B and S2D). However, ras1G12V;csk−/−-mediated upregulation of MMP1 expression was independent of dietary sugar (Figures 2E and 2F). Our data suggest that HDS enhances breakdown of the basement membrane to facilitate spreading and metastasis-like behavior of transformed cells, at least in part by mechanisms independent of MMP1 expression levels.
- Deryugina E.I.
- Quigley J.P.
Matrix metalloproteinases and tumor metastasis.
Cancer Metastasis Rev. 2006; 25: 9-34
Larvae fed HDS consistently (93.0%) displayed GFP-labeled ras1G12V;csk−/− cells scattered throughout their hemolymph, a blood analog (Figures 2I–2K and 2S). A total of 19.1% of these larvae also developed secondary rasG12V;csk−/− tumors distant from the eye field that continued to proliferate (Figures 2L, 2P, and 2S); secondary tumors were rarely (0.8%) observed in animals fed control food (Figure 2S). Whereas the percentage of animals with loss of Laminin in the eye tissue correlated with the percentage of animals with GFP-positive cells in the hemolymph, readily scorable secondary tumors were found in only a subset (19.1%) of the animals fed HDS (Figure 2S).
Combining ras1G12V with a weaker csk allele, cskj1D8 (ras1G12V;cskhypo/hypo), led to a reduced penetrance of secondary tumors (Figures S2E, S2F, S2F′, and S2G), suggesting that levels of Src activity play a primary role in distant Ras/Src tumor formation. We also examined scribbled, which encodes a protein required to maintain apical/basal cell polarity (
Bilder and Perrimon, 2000). As previously reported (
- Bilder D.
- Perrimon N.
Localization of apical epithelial determinants by the basolateral PDZ protein Scribble.
Nature. 2000; 403: 676-680
Pagliarini and Xu, 2003;
- Pagliarini R.A.
- Xu T.
A genetic screen in Drosophila for metastatic behavior.
Science. 2003; 302: 1227-1231
Brumby and Richardson, 2003), targeting ras1G12V;scrib−/− to the eye led to substantial overgrowth; however, we did not observe secondary tumors in these animals when fed either a control diet or HDS (Figures S2H–S2J). These results indicate a specific relationship between Ras and Csk/Src activities that, when combined with HDS, synergize to produce secondary tumors. The data also indicate that tumor overgrowth and secondary tumor formation are separable processes and, similar to mammals (
- Brumby A.
- Richardson H.
scribble mutants cooperate with oncogenic Ras or Notch to cause neoplastic overgrowth in Drosophila.
Mehlen and Puisieux, 2006), only a small subset of circulating cells successfully seed a secondary tumor.
- Mehlen P.
- Puisieux A.
Metastasis: a question of life or death.
Nat. Rev. Cancer. 2006; 6: 449-458
Larger secondary ras1G12V;csk−/− tumors were typically tightly associated with the tracheal system, a tubular network that transports oxygen (Figures 2L–2O). A Chitin-binding probe (
Devine et al., 2005) indicated that secondary tumors enwrapped tracheal branches (Figure 2Q). Interestingly, Chitin was abnormally deposited within these tumor-imbedded trachea (Figure 2Q), suggesting either tracheal alteration or de novo branching. Consistent, but not proving the latter view, secondary ras1G12V;csk−/− tumors expressed Branchless (Figure 2R), a fly fibroblast growth factor (FGF) ortholog capable of promoting de novo trachea branching (
- Devine W.P.
- Lubarsky B.
- Shaw K.
- Luschnig S.
- Messina L.
- Krasnow M.A.
Requirement for chitin biosynthesis in epithelial tube morphogenesis.
Proc. Natl. Acad. Sci. USA. 2005; 102: 17014-17019
Sutherland et al., 1996). Together, these results indicate that HDS enhances or establishes multiple aspects of tumorigenesis in the presence of activated Ras/Src, including proliferation, a block in apoptosis, invasive migration, and establishment of secondary tumors distant from the primary tumor.
- Sutherland D.
- Samakovlis C.
- Krasnow M.A.
branchless encodes a Drosophila FGF homolog that controls tracheal cell migration and the pattern of branching.
Cell. 1996; 87: 1091-1101
The PI3K Pathway Mediates Diet-Induced Tumorigenesis
Genetically reducing PI3K activity through a hypomorphic allele of akt (ras1G12V;csk−/−,akthypo/hypo; Figure 3B) or through feeding of the PI3K inhibitor wortmannin (Figure S3) led to strongly reduced tumor cell survival in the presence of HDS. The Tor-class inhibitor rapamycin displayed a similar reduction in tumor tissue (Figure S3); in addition, animals were significantly smaller with an extended third-instar larval stage, as previously reported (
Zhang et al., 2000). Conversely, coexpressing the constitutively active insulin receptor isoform inRCA (inRCA,ras1G12V;csk−/−) in animals fed a control diet prevented elimination of clones from the epithelia. The result was tumor overgrowth in which 91.0% of larvae displayed an enlarged eye disc phenotype (Figures 3D and 3F). inRCA,ras1G12V;csk−/− animals did not exhibit any further increase in primary tumor size when raised on HDS (Figure 3E), nor did they exhibit enhanced tumors when compared to ras1G12V;csk−/− in HDS (compare Figures 1J and 3F). We conclude that components of the Insulin/PI3K pathway are required autonomously within the ras1G12V;csk−/−-transformed cells for enhancement by HDS.
- Zhang H.
- Stallock J.P.
- Ng J.C.
- Reinhard C.
- Neufeld T.P.
Regulation of cellular growth by the Drosophila target of rapamycin dTOR.
Genes Dev. 2000; 14: 2712-2724
Of note, Insulin/PI3K signaling did not mediate all of the effects of HDS. While the triple combination inRCA,ras1G12V;csk−/− fed a control diet exhibited strongly enhanced expansion of tumors within the eye field, overgrowth was not accompanied by metastasis-like behavior or secondary tumor formation. Further, though elevated MMP1 expression was observed, the basement membrane was still retained (Figures 2C and 2G). This result again emphasizes that tumor overgrowth and metastasis are separable processes.
Ras/Src-Activated Cells Remain Sensitive to Insulin and Glucose
Our results suggest that insulin signaling is required to mediate HDS-enhanced growth. However, eye epithelial tissue in control animals raised on HDS exhibited reduced phosphorylated, activated Akt upon Insulin stimulation, an indication of insulin resistance as previously reported (Figure 3G) (
Musselman et al., 2011). This led us to a central question in tumor metabolism studies: how does a diet that promotes insulin resistance also enhance tumors in an Insulin/PI3K-dependent manner?
- Musselman L.P.
- Fink J.L.
- Narzinski K.
- Ramachandran P.V.
- Hathiramani S.S.
- Cagan R.L.
- Baranski T.J.
A high-sugar diet produces obesity and insulin resistance in wild-type Drosophila.
Dis. Model. Mech. 2011; 4: 842-849
In contrast to controls, eye tissue within ras1G12V;csk−/− animals fed HDS exhibited an increase in baseline Akt when compared to control-diet-fed animals (Figure 3H), suggesting these tissues were in fact not insulin resistant. Consistent with this view, challenging eye tissue from HDS animals with exogenous Insulin led to a strong increase in phospho-Akt (Figure 3H). Insulin-stimulated increase in phospho-Akt was observed autonomously within the ras1G12V;csk−/− clones (Figure 3I). These data indicate that activating the Ras and Src pathways reversed HDS-induced insulin resistance, leading to hypersensitive responsiveness to Insulin. The result was increased sugar flux; in the presence or absence of exogenous Insulin, ras1G12V;csk−/− animals raised on HDS displayed a significant increase in glucose uptake to a level comparable to the triple combination inRCA,ras1G12V;csk−/− (Figures 3J–3N). Reducing akt activity (ras1G12V;csk−/−,akthypo/hypo) blocked glucose uptake (Figure 3O). We conclude that ras1G12V;csk−/− cells escape diet-induced insulin resistance to utilize both increased insulin responsiveness and insulin-pathway-dependent glucose uptake. We next explored the mechanisms by which transformed cells evade insulin resistance.
Tumors Exhibit Aspects of an "Undead" Phenotype
A central hallmark of mammalian cancer is the ability of transformed cells to evade apoptosis. In Drosophila developing eye and wing epithelia, keeping otherwise apoptotic cells alive (undead) by blocking effector caspase activity leads to aberrant tissue overgrowth due to compensatory proliferation of surrounding cells, a process designed to maintain tissue size and integrity (reviewed in
Bergmann and Steller, 2010,
- Bergmann A.
- Steller H.
Apoptosis, stem cells, and tissue regeneration.
Sci. Signal. 2010; 3: re8
Fan and Bergmann, 2008,
- Fan Y.
- Bergmann A.
Apoptosis-induced compensatory proliferation. The Cell is dead. Long live the Cell!.
Trends Cell Biol. 2008; 18: 467-473
Martín et al., 2009). We used an antibody that recognizes the cleaved, active isoform of human caspase-3, a validated readout for activity of the Drosophila initiator caspase Dronc (
- Martín F.A.
- Peréz-Garijo A.
- Morata G.
Apoptosis in Drosophila: compensatory proliferation and undead cells.
Int. J. Dev. Biol. 2009; 53: 1341-1347
Fan and Bergmann, 2010). We observed broad Dronc activity in ras1G12V;csk−/− clones of animals raised on a control diet (Figures 4A and S4A). A significant proportion of these cells were undergoing apoptotic cell death as assessed by TUNEL staining (Figures 4D and S4A).
- Fan Y.
- Bergmann A.
The cleaved-Caspase-3 antibody is a marker of Caspase-9-like DRONC activity in Drosophila.
Cell Death Differ. 2010; 17: 534-539
In the presence of HDS, ras1G12V;csk−/− clones also exhibited broad cytosolic Dronc activity (Figures 4B and S4A). However, these clones were almost completely negative for apoptotic cell death as assessed by TUNEL and DAPI analysis (Figures 4E and S4A; data not shown); a few cells outside of the clone region were TUNEL positive. Insulin pathway activation alone was sufficient to mimic this block: inRCA,ras1G12V;csk−/− animals fed a control diet retained Dronc caspase activity but, again, were resistant to apoptotic cell death as assessed by TUNEL (Figures 4C, 4F, and S4A). One candidate to block apoptosis in transformed cells is the caspase inhibitor Drosophila inhibitor of apoptosis protein 1 (Diap1), which inhibits caspases downstream of Dronc (
Ditzel et al., 2008). diap1 gene expression and Diap1 protein levels were strongly increased in the ras1G12V;csk−/− clones of animals raised in HDS compared to animals fed a control diet (Figures 4G–4I, S4B, and S4C). The triple combination inRCA,ras1G12V;csk−/− was sufficient to elevate diap1 expression and Diap1 protein levels in animals fed a control diet, whereas reduced akt activity (ras1G12V;csk−/−,akthypo/hypo) failed to elevate Diap1 in HDS animals (Figures 4J, S4D, and S4E). These results indicate that elevation of insulin pathway signaling through HDS leads to elevated Diap1 and a block in apoptotic cell death downstream of cleaved, activated Dronc. Interestingly, secondary tumors retained diap1 expression but lost Dronc activity (Figures S4G and S4H).
- Ditzel M.
- Broemer M.
- Tenev T.
- Bolduc C.
- Lee T.V.
- Rigbolt K.T.
- Elliott R.
- Zvelebil M.
- Blagoev B.
- Bergmann A.
- Meier P.
Inactivation of effector caspases through nondegradative polyubiquitylation.
Mol. Cell. 2008; 32: 540-553
Increased Wingless/Wnt Signaling Mediates Diet-Induced Tumorigenesis
Previous work in Drosophila compensatory proliferation found that undead cells also elevate expression of the Wnt ortholog Wingless (Wg); this mitogenic signal in turn directs local hyperplasia (
Pérez-Garijo et al., 2009). In ras1G12V;csk−/− eye clones, we detected low levels of Wg protein within the clones in animals fed a control diet (Figure 4K). Notably, Wg protein levels were strongly upregulated in animals fed HDS (Figures 4L and 4M). Reducing akt activity (ras1G12V;csk−/−,akthypo/hypo) prevented this elevation (Figure S4F), whereas the triple combination inRCA,ras1G12V;csk−/− displayed increased Wg levels in animals fed a control diet (Figure 4N). Activation of InR alone was not sufficient to elevate Wg levels (Figure S5A). We conclude that combining elevated InR, Ras, and Src activities leads to elevated Wg expression in discrete tumors.
- Pérez-Garijo A.
- Shlevkov E.
- Morata G.
The role of Dpp and Wg in compensatory proliferation and in the formation of hyperplastic overgrowths caused by apoptotic cells in the Drosophila wing disc.
Development. 2009; 136: 1169-1177
Elevation of Wg contributed to tumor progression. Reducing Wg activity (ras1G12V;csk−/−,wgRNAi) in animals fed HDS led to strong suppression of tumor growth (Figure 5B). Expression of a dominant-negative isoform of the downstream pathway effector Tcf (ras1G12V;csk−/−,tcfDN) also suppressed tumor growth (Figure 5C), indicating that canonical Wg signaling is required for high-sucrose-induced enhancement of tumorigenesis. As a result, whereas only 22.5% of ras1G12V;csk−/− animals successfully initiated pupariation when fed HDS, 100% of ras1G12V;csk−/−,wgRNAi or ras1G12V;csk−/−,tcfDN animals successfully pupariated when fed HDS (Figures S5B and S5C).
In undead cells, Jnk pathway signaling is required for ectopic Wg expression (
Ryoo et al., 2004). Expressing a dominant-negative isoform of the Drosophila Jnk ortholog Basket bskDN (ras1G12V;csk−/−,bskDN) suppressed tumor growth in HDS (Figures 5D and S5D) and blocked the elevation in Wg protein levels (Figure 5E), indicating that Wg upregulation is downstream of Bsk. Wg was also blocked in inRCA,ras1G12V;csk−/−,bskDN eye clones of animals fed a control diet, indicating Bsk is in turn downstream of InR with regard to elevated Wg (Figure 5F). Together, these results indicate that ras1G12V;csk−/− cells achieve an undead status through Diap1, which then activates a InR-Jnk-Wg pathway that promotes cell-autonomous proliferation. We next asked whether this ability of Wg to mediate key aspects of HDS/insulin pathway activity within Ras/Src-activated tumors extended to evasion of insulin resistance.
- Ryoo H.D.
- Gorenc T.
- Steller H.
Apoptotic cells can induce compensatory cell proliferation through the JNK and the Wingless signaling pathways.
Dev. Cell. 2004; 7: 491-501
Canonical Wingless/Wnt Pathway Signaling Promotes Insulin Sensitivity by Regulating InR Expression
In organ culture experiments from animals fed HDS, challenging control tissue with exogenous Insulin led to little or no increase in pathway activity as assessed by phosphorylated Akt (Figure 5G); we also observed significant downregulation of inR messenger RNA (mRNA) (Figure 5H), which presumably at least partially accounts for the tissues' insulin resistance. Two other members of the Insulin pathway, Chico/IRS and Lnk/SH2B, showed no change in transcript levels (Figure 5H), suggesting that diet-directed downregulation is specific to InR. In contrast, tissue expressing Wg responded to exogenous Insulin by strongly activating pathway signaling (Figure 5G). This suggests that, in addition to Insulin regulating Wg, Wg regulates Insulin signaling, potentially through the regulation of inR expression.
Consistent with this model, inR mRNA levels were significantly upregulated in ras1G12V;csk−/− eye clones in animals fed HDS (Figure 5I). The result was an increase in phosphorylated, activated InR that was strongly enhanced when the tissue was challenged with exogenous Insulin (Figure 5J). Importantly, the diet-dependent increase in inR mRNA levels was suppressed by reducing activity of the Wg target Tcf (ras1G12V;csk−/−,tcfDN; Figure 5I). These results indicate that canonical Wg signaling promotes insulin sensitivity by upregulating inR transcripts. That is, transformed tissue evaded insulin resistance through a feed-forward mechanism by which elevated Wg directs elevated InR (Figure 5K).
Combinatorial Multinode Drug Treatments for Diet-Induced Ras/Src Tumorigenesis
Identifying an HDS-dependent undead cell phenotype in ras1G12V;csk−/− tissue suggested specific points of therapeutic intervention: (1) conversion of dietary sucrose, (2) InR/PI3K metabolic signaling, (3) canonical Wg/Wnt pathway undead cell signaling, and (4) Ras plus Src oncogenic pathways (Figure 5K). Nearly three-quarters of ras1G12V;csk−/− animals raised on HDS failed to pupariate due to aggressive eye tumors; we utilized pupariation rate as a quantitative viability assay to take a stepwise approach toward identifying an optimized whole-animal therapeutic cocktail. Time to pupariation was not affected by drug treatments when compared to vehicle (DMSO) treatment alone; pupariation rates were assessed at day 17 AEL, after pupariation curves reached a plateau (Figure S6A).
Acarbose is an inhibitor of α-glucosidase that is used to treat type 2 diabetes mellitus (
Anderson, 2005). Feeding acarbose in the presence of HDS led to reduced tumor growth and improved ras1G12V;csk−/− animal survival (Figures 6B and 6J ). Pyrvinium, a small-molecule inhibitor of the canonical Wnt-signaling pathway (
- Anderson Jr., D.C.
Pharmacologic prevention or delay of type 2 diabetes mellitus.
Ann. Pharmacother. 2005; 39: 102-109
Thorne et al., 2010), suppressed HDS-induced tumor growth at a concentration that did not affect the growth of wild-type clones (Figures 6C and S6B); 41.9% of HDS- and pyrvinium-fed animals achieved pupariation (Figure 6J). We recently developed polypharmacological anticancer compounds (
- Thorne C.A.
- Hanson A.J.
- Schneider J.
- Tahinci E.
- Orton D.
- Cselenyi C.S.
- Jernigan K.K.
- Meyers K.C.
- Hang B.I.
- Waterson A.G.
- et al.
Small-molecule inhibition of Wnt signaling through activation of casein kinase 1α.
Nat. Chem. Biol. 2010; 6: 829-836
Dar et al., 2012) that target the Ras, Src, and Tor pathways as single agents. These represent the central cellular pathways that mediate HDS-induced tumor growth in ras1G12V;csk−/− animals and, indeed, the compound AD81 suppressed Ras/Src-tumorigenesis and larval lethality of animals fed HDS (Figures 6D and 6J). Biochemical analysis of dissected eye tissues supported inhibition of Ras, Src, and Tor pathways by AD81 feeding, though not by the close analog AD80 (Figure 6I) (
- Dar A.C.
- Das T.K.
- Shokat K.M.
- Cagan R.L.
Chemical genetic discovery of targets and anti-targets for cancer polypharmacology.
Nature. 2012; 486: 80-84
Dar et al., 2012). Furthermore, AD81 also suppressed tumor growth and larval lethality of inRCA,ras1G12V;csk−/− animals fed a control diet, demonstrating AD81 can act tumor autonomously (Figure S6C).
- Dar A.C.
- Das T.K.
- Shokat K.M.
- Cagan R.L.
Chemical genetic discovery of targets and anti-targets for cancer polypharmacology.
Nature. 2012; 486: 80-84
Targeting sugar or the Ras, Src, PI3K, or Wg pathways showed significant but limited efficacy on ras1G12V;csk−/− tumors in animals fed HDS. Combining acarbose with pyrvinium, acarbose with AD81, or pyrvinium with AD81 enhanced survival to permit 64.4%, 62.9%, and 68.5%, survival of ras1G12V;csk−/− animals to pupariation in the presence of HDS, respectively (Figures 6E–6G and 6J). Strikingly, the triple combination acarbose-pyrvinium-AD81 achieved a 91.2% rescue level of ras1G12V;csk−/− animals fed HDS (Figures 6H and 6J), far higher than any single or double drug combination we have tested. We conclude that targeting multiple nodes in a rational manner may be required to achieve an optimal therapeutic index, balancing efficacy with minimal whole-animal toxicity.
Epidemiological studies provide strong evidence for an association between metabolism-related diseases and cancer, but the underlying mechanisms that link these two diseases remain poorly understood. In this study, we demonstrate that dietary sucrose strongly enhances multiple aspects of Ras/Src-activated tumors in Drosophila. HDS led to stabilization of tumors within the epithelium, increased proliferation, and metastasis-like behavior. Mechanistically, we provide evidence that Ras/Src-activated cells increase insulin pathway sensitivity in an otherwise insulin-resistant environment, allowing them to take advantage of high circulating glucose. The result is a Wg- and Jnk-dependent enhancement of tumor progression. Our results support a model in which Ras/Src cells become hypersensitive to circulating Insulin, leading to efficient activation of insulin/PI3K signaling (Figure 5K).
A key consequence of increased insulin/PI3K signaling was the prevention of apoptosis and the promotion of elevated levels of Wg expression and activity; these two aspects promoted an undead-like cell phenotype that further enhanced proliferation. We observed low but detectable levels of Wg expression in ras1G12V;csk−/− clones in animals fed a control diet (Figure 4K); these clones exhibited high levels of apoptosis. In contrast, Wg accumulated at significantly higher levels in ras1G12V;csk−/− tumors within animals fed HDS (Figure 4L). High Wg levels led to elevated Diap1 expression and reduced apoptosis, stabilizing ras1G12V;csk−/− clones and revealing accumulation of Wg within the clone. Our model suggests that inhibition of cell death in "HDS tumors" is a key aspect of Ras/Src-mediated transformation and may allow cells to stably and continuously produce Wg. Wg was expressed within ras1G12V;csk−/− cells in the presence of HDS (Figure 4L and 4M), and phosphorylated Akt levels increased within Ras/Src clones, but not detectably in nearby wild-type cells (Figure 3I). Therefore, unlike the conventional undead situation (induced by Hid plus P35), a Wg-mediated increase in InR expression and signaling provides a growth advantage specifically within Ras/Src cells rather than surrounding wild-type tissue. Together, our data provide evidence that the undead cell phenotype generates an InR-Jnk-Wg-InR feed-forward mechanism that results in elevated InR expression and a reversal of insulin resistance (Figure 5K). The result is transformed tissue that is optimized for insulin-dependent glucose uptake in an animal with hyperinsulinemia and hyperglycemia.
The undead phenotype was not fully maintained in secondary tumors; Dronc expression was lost in distant tumors of ras1G12V;csk−/−, though diap1 expression was maintained (Figures S4G and S4H). These results suggest that secondary tumors develop progressively distinct properties; understanding these changes will require additional studies. A Chitin-binding probe suggested progressive steps of tubule maturation within secondary tumor-associated trachea, including potentially newly expressed Chitin scattered within immature tubules, tubules filled with solid projections of Chitin, and hollowed out mature tubules (Figure 2Q). Additional studies will be required to confirm that this represents neotracheogenesis and, if so, whether this occurs through processes reminiscent of the neoangiogenesis that is a common feature of mammalian metastasis (
Bergers and Benjamin, 2003). Strikingly, secondary tumors expressed high levels of the FGF ortholog Branchless, an important step in promoting tracheogenesis during development (Figure 2R;
- Bergers G.
- Benjamin L.E.
Tumorigenesis and the angiogenic switch.
Nat. Rev. Cancer. 2003; 3: 401-410
Sutherland et al., 1996).
- Sutherland D.
- Samakovlis C.
- Krasnow M.A.
branchless encodes a Drosophila FGF homolog that controls tracheal cell migration and the pattern of branching.
Cell. 1996; 87: 1091-1101
The Drosophila Wnt ortholog Wg promotes a tumor's ability to evade insulin resistance through its regulation of inR expression in the presence of HDS feeding, which otherwise promotes diabetes-like phenotypes (Figure 5H) (
Musselman et al., 2011). High Wg expression requires both Ras/Src activation and HDS, promoting inR expression through canonical Wg-dTcf signaling (Figure 5I). These results emphasize a link between canonical Wg signaling and Insulin signaling. Consistent with this model linking canonical Wg and Insulin signaling pathways, a recent study reports that reduced expression of skeletal muscle Insulin receptor, which is linked to hyperinsulinemia and insulin resistance, is enhanced by a mutation in the Wnt coreceptor LRP6 in humans (
- Musselman L.P.
- Fink J.L.
- Narzinski K.
- Ramachandran P.V.
- Hathiramani S.S.
- Cagan R.L.
- Baranski T.J.
A high-sugar diet produces obesity and insulin resistance in wild-type Drosophila.
Dis. Model. Mech. 2011; 4: 842-849
Singh et al., 2013).
- Singh R.
- De Aguiar R.B.
- Naik S.
- Mani S.
- Ostadsharif K.
- Wencker D.
- Sotoudeh M.
- Malekzadeh R.
- Sherwin R.S.
- Mani A.
LRP6 enhances glucose metabolism by promoting TCF7L2-dependent insulin receptor expression and IGF receptor stabilization in humans.
Cell Metab. 2013; 17: 197-209
Tumors with Wnt activation may represent tumor types that respond and synergize with metabolic defects. Wnt activation is frequently observed in many tumor types, including those with a strong association with diabetes, such as hepatocellular carcinomas and colorectal cancers; activating mutations in β-catenin and loss-of-function mutations in APC are frequently found in human hepatocellular carcinoma and colorectal cancers (
Fodde et al., 2001,
- Fodde R.
- Smits R.
- Clevers H.
APC, signal transduction and genetic instability in colorectal cancer.
Nat. Rev. Cancer. 2001; 1: 55-67
Laurent-Puig and Zucman-Rossi, 2006). Incidence of pancreatic cancer has a particularly strong association with diabetes, suggesting that its constituent mutation load may interface with metabolic dysfunction. Activating mutations in K-Ras plus upregulation of Src activity are commonly found in pancreatic ductal adenocarcinoma (PDA) (
- Laurent-Puig P.
- Zucman-Rossi J.
Genetics of hepatocellular tumors.
Oncogene. 2006; 25: 3778-3786
Almoguera et al., 1988,
- Almoguera C.
- Shibata D.
- Forrester K.
- Martin J.
- Arnheim N.
- Perucho M.
Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes.
Cell. 1988; 53: 549-554
Morton et al., 2010), and a mouse K-Ras/Src pancreatic model developed PDA with shortened latency (
- Morton J.P.
- Karim S.A.
- Graham K.
- Timpson P.
- Jamieson N.
- Athineos D.
- Doyle B.
- McKay C.
- Heung M.Y.
- Oien K.A.
- et al.
Dasatinib inhibits the development of metastases in a mouse model of pancreatic ductal adenocarcinoma.
Gastroenterology. 2010; 139: 292-303
Shields et al., 2011). Wnt ligands and transcriptional target genes of β-catenin are highly expressed in PDA (
- Shields D.J.
- Murphy E.A.
- Desgrosellier J.S.
- Mielgo A.
- Lau S.K.
- Barnes L.A.
- Lesperance J.
- Huang M.
- Schmedt C.
- Tarin D.
- et al.
Oncogenic Ras/Src cooperativity in pancreatic neoplasia.
Oncogene. 2011; 30: 2123-2134
Pasca di Magliano et al., 2007), a phenomenon reminiscent of our observations linking diet, Ras/Src, and Wnt signaling. A clear link has yet to be established between pancreatic cancers featuring elevated Ras/Src activity and/or Wnt-pathway activation within diabetic patients.
- Pasca di Magliano M.
- Biankin A.V.
- Heiser P.W.
- Cano D.A.
- Gutierrez P.J.
- Deramaudt T.
- Segara D.
- Dawson A.C.
- Kench J.G.
- Henshall S.M.
- et al.
Common activation of canonical Wnt signaling in pancreatic adenocarcinoma.
PLoS ONE. 2007; 2: e1155
Our data indicate that limiting dietary sucrose directly or through blocking its intermediates through compounds such as acarbose may reduce important aspects of tumor risk and progression in individuals with metabolism-related diseases. Combining acarbose with drugs that target the triggering Ras/Src pathways plus components of the amplification circuit provided the most robust therapeutic impact on diet-enhanced tumors in our model. These results provide a potential explanation for how insulin-resistant animals are at increased risk for tumorigenesis and emphasize the importance of targeting multiple, specific nodes to achieve optimal therapeutic value.
Experimental Procedures
Fly Stocks
UAS-ras1G12V, UAS-inRA1325D (inRCA), akt04226, cskj1D8, UAS-tcfDN, UAS-bskDN, thj5c8 (diap1-lacZ), and UAS-HA-wg flies were obtained from the Bloomington Stock Center. UAS-wgRNAi flies were obtained from the Vienna Drosophila RNAi Center. The following stocks were kindly provided to us: FRT82B, cskQ156Stop (by A. O'Reilly and M. Simon), scrib1 (by D. Bilder), and ey(3.5)-FLP1 (by G. Halder).
To create eyeless-driven GFP-labeled clones, flies with the genotype ey(3.5)-FLP1; act > y+ > gal4,UAS-GFP; FRT82B, tub-gal80 were crossed with flies with the following genotypes: (1) UAS-lacZ; FRT82B; (2) UAS-lacZ; FRT82B, cskQ156Stop/TM6b; (3) UAS-ras1G12V; FRT82B; (4) UAS-ras1G12V; FRT82B, cskQ156Stop/TM6b; (5) UAS-ras1G12V; FRT82B, cskj1D8/TM6b; (6) UAS-ras1G12V; FRT82B, scrib1/TM6b; (7) UAS-inRA1325D, UAS-ras1G12V; FRT82B, cskQ156Stop/TM6b; (8) UAS-ras1G12V; FRT82B, cskQ156Stop, akt04226/TM6b; (9) UAS-ras1G12V; FRT82B, cskQ156Stop, UAS-wgRNAi/TM6b; (10) UAS-ras1G12V; FRT82B, cskQ156Stop, UAS-tcfDN/TM6b; (11) UAS-ras1G12V; FRT82B, cskQ156Stop, UAS-bskDN/TM6b; (12) UAS-inRA1325D, UAS-ras1G12V; FRT82B, cskQ156Stop, UAS-bskDN/TM6b; (13) UAS-inRA1325D; FRT82B; (14) FRT82B, UAS-HA-wg; and (15) UAS-ras1G12V; diap1-lacZ, FRT82B, cskQ156Stop/TM6b; (16) UAS-inrA1325D, UAS-ras1G12V; diap1-lacZ, FRT82B, cskQ156Stop/TM6b.
Cultures were carried out on Bloomington semidefined medium (described by the Bloomington Stock Center) with modifications. Detailed recipes for the control diet, HDS, and the high-fat diet have been previously described (
Musselman et al., 2011). The following final concentrations of carbohydrates included 0.15 M sucrose (control diet), 1.0 M sucrose (HDS), and 2.0 M glucose (high-glucose diet). Cultures were performed at 25°C unless otherwise noted.
- Musselman L.P.
- Fink J.L.
- Narzinski K.
- Ramachandran P.V.
- Hathiramani S.S.
- Cagan R.L.
- Baranski T.J.
A high-sugar diet produces obesity and insulin resistance in wild-type Drosophila.
Dis. Model. Mech. 2011; 4: 842-849
Immunofluorescence and TUNEL Labeling
Larval eyes were dissected in PBS, fixed in 4% paraformaldehyde (PFA) in PBS or PLP-fixative (2.5% PFA, 0.075 M lysine, 0.25% [w/v] Na-periodate in phosphate buffer), washed in PBT (PBS containing 0.1% Triton X-100), and incubated with primary antibodies in PAXDG (PBS containing 1% BSA, 0.3% Triton X-100, 0.3% deoxycholate, and 5% goat serum), followed by washing and incubation with secondary antibodies in PAXDG. Tissues were counterstained with TO-PRO-3 (Invitrogen) and mounted in Vectashield mounting media (Vector Laboratories). Antibodies used were rabbit anti-cleaved caspase-3 (Asp175; Cell Signaling), mouse anti-dMMP1 (Developmental Studies Hybridoma Bank [DSHB]), mouse anti-Wingless (DSHB), mouse anti-β-galactosidase (DSHB), rabbit anti-Laminin β 1 (Abcam), rabbit anti-phospho-histone H3 (Ser10; Millipore), rabbit anti-Diap1 (a gift from H. Steller), rabbit anti-phospho-Akt (Ser473; Cell Signaling), and rat anti-Branchless (a gift from M. Krasnow). Secondary Alexa-568-conjugated anti-mouse and anti-rabbit antibodies were used (Molecular Probes). To label tracheal lumen, Rhodamine-conjugated Chitin-binding probe was used (New England Biolabs).
TUNEL was performed using the TMR-Red In Situ Cell Detection Kit (Roche Diagnostics). Samples were fixed for 15 min in 4% PFA in PBT, washed in PBT and permeabilized in 100 mM sodium citrate/0.1% Triton X-100 at 65°C for 30 min, washed in PBT, and free DNA ends were fluorescein labeled.
Fluorescent and confocal images were taken with Leica DM5500 microscope equipped with DFC340 FX Monochrome Digital Camera and TSC SPE confocal microscope.
Compounds solubilized in DMSO were diluted directly into the fly medium and vortexed extensively to obtain homogeneous culture. Drugs used were wortmannin and rapamycin (LC Laboratories) and acarbose and pyrvinium pamoate (Sigma). AD compounds were provided by A. Dar and K. Shokat (UCSF).
Western Blotting
Wandering third-instar larvae were rinsed in PBS; the eye discs were dissected out and placed in Schneider's Drosophila medium on ice. The eye discs were incubated with or without 5 μM recombinant human Insulin (Sigma) for 15 min at room temperature. Eye discs were transferred to a microcentrifuge tube and spun down briefly. The supernatant was discarded and the remaining precipitates were suspended in SDS sample buffer at a concentration of 10 μl/mg tissue and used to generate western blots. Antibodies used targeted total Akt (Cell Signaling), phospho-Drosophila Akt (Ser505; Cell Signaling), phospho-IGF-1 receptor β (Tyr1131)/Insulin receptor β (Tyr1146; Cell Signaling), and Syntaxin (DSHB). Secondary antibodies were from Cell Signaling.
Glucose Uptake Assay
For the glucose uptake experiments, flies with the genotype ey(3.5) FLP1; act > y+ > gal4; FRT82B, tub-gal80 were crossed with flies with the following genotypes: (1) UAS-lacZ; FRT82B; (2) UAS-ras1G12V; FRT82B, cskQ156Stop/TM6b; (3) UAS-inRA1325D, UAS-ras1G12V; FRT82B, cskQ156Stop/TM6b; and (4) UAS-ras1G12V; FRT82B, cskQ156Stop, akt04226/TM6b.
Wandering third-instar larvae were rinsed in PBS; the eye discs were dissected out and placed in Drosophila larval saline (HEPES-NaOH [pH 7.1], 87 mM NaCl, 40 mM KCl, 8 mM CaCl2, 8 mM MgCl2, 50 mM sucrose, and 5 mM trehalose) on ice. The eye discs were incubated with or without 5 μM recombinant human Insulin (Sigma) in the presence of 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG: Invitrogen) for 15 min at room temperature. The tissues were washed with Drosophila larval saline and mounted with a Vectashield (Vector Laboratories).
Quantitative RT-PCR
Total RNA was extracted from the eye discs using the QIAGEN RNeasy Mini Kit. Oligo-dT primers and Superscript RT-II reverse transcriptase (Invitrogen) was used to synthesize first-strand DNA. PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems) and analyzed on the Applied Biosystems StepOnePlus real-time PCR system. Results were normalized to Kinesin mRNA, and RpL32 was used as a control. The following primers were used: Kinesin-f, 5′-GCTGGACTTCGGTCGTAGAG-3′; Kinesin-r, 5′-CTTTTCATAGCGTCGCTTCC-3′; RpL32-f, 5′- GCTAAGCTGTCGCACAAATG-3′; RpL32-r, 5′-GTTCGATCCGTAACCGATGT-3′; InR-f, 5′- ACAAAATGTAAAACCTTGCAAATCC-3′; InR-r, 5′-GCAGGAAGCCCTCGATGA-3′; chico-f, 5′-ATCAGGCGATGCGGTC-3′; chico-r, 5′-ACATAGCGCTCAGTATCG-3′; dLnk-f, 5′-GTGTTCGCCTAAAGACAAATGAAAT-3′; dLnk-r, 5′-TGTTAGCGTTGTGGGATCCAA-3′.
We thank J. Diaz, J. Na, J. Pendse, L.P. Musselman, J. Fink, M. Vidal, and current and former Baranski and Cagan lab members for helpful discussions; C.Y. Nien for writing MATLAB scripts for quantitation; Y. Tomer and D. LeRoith for manuscript comments; and A. O'Reilly, M. Simon, D. Bilder, G. Halder, H. Steller, M. Krasnow, A. Dar, and K. Shokat for reagents. We also thank the Bloomington Stock Center, the Vienna Drosophila RNAi Center, and the Developmental Studies Hybridoma Bank for fly strains and antibodies. This research was supported by the National Cancer Institute (grants R01 CA170495 and R01 CA109730 to S.H. and R.L.C. and grant R21 DK069940 to R.L.C. and T.J.B.). R.L.C. and T.J.B. are cofounders of Medros, which uses fly models of human disease for drug development.
Supplemental Information
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Article Info
Publication History
Published: August 1, 2013
Accepted: June 19, 2013
Received in revised form: May 8, 2013
Received: November 17, 2012
© 2013 Elsevier Inc. Published by Elsevier Inc.
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text:
Padraig wants to 'win, win, win'
Padraig Harrington will return to competitive action this week knowing that he needs to return to the winner's circle if he is to qualify for the Masters.
The 42-year-old's victory drought stretches back to August 2008 when he won the PGA Championship just a few months after claiming the British Open at Royal Birkdale.
His poor form has seen him slip to number 130 in the world rankings and his five-year exemption to the Masters that he earned with his two major titles in 2008 has also expired.
It means he needs to return to top 50 to gain an automatic invitation to Augusta National in April and the only way to do that is to start winning again.
"Win, win, win," Harrington said ahead of the Phoenix Open. "That's pretty straight forward for me."
He added: "It's a long shot, you know, to get into the Masters. First tournament of the year is always a great tournament. You never know what you're going to get. It's always nice your first week out."
One thing to look forward to at TPC Scottsdale is the enthusiastic fans and the famous par-three 16th, the only fully enclosed hole on the PGA Tour, likely to be the place to be once again.
"I don't think you can describe it to people," Harrington said. "And I do believe that every person reacts a little bit different to it … For golf, there is nothing like it."
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subset: commoncrawl


text:
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Omicron has affected everyone, even the young kids as well. Hospitals around the nation are now seeing an increase in kids cases which was not present during the last surge with delta or any other variant.
How To Safeguard The Kids From Omicron Variant?
This has caused some panic among the parents as they are not sure if they should be sending the kids to school or for any outdoor activities.
Most of the schools in the US are now turning to remote learning at least for a week and have asked all the students to get themselves tested during this period. Many of the schools have also made it mandatory that the students present a negative certificate when they come over for in-person learning from next week.
Dr. Leana Wen, who is an emergency physician from George Washington University of Public health, stated that parents now have many questions in regards to their children continuing remote learning.
Wen stated that many parents and children have stated that in-person learning is more effective as this helps them to understand the concepts better; however, seeing the current situation, it is better for the parents to ask the schools to be in remote learning rather than getting their kids infected.
Few of the parents stated that they feel their kids won't pick up the necessary social skills which are required in the real world with remote learning.
Dr. Wen replied to this, stating that this may be true in many senses; however, it is now being proved that kids are connecting to many other people of their same age through virtual networks, thereby breaking the bonds of geographical restrictions as well.
This helps them understand different cultures at the same time and also helps them to bond with people from different nationalities. Wen also mentioned that social skills are a necessity, and parents should interact with the kids on a daily basis so that they know what the basic etiquettes are while speaking to an adult or how to stay silent while someone else is speaking.
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In this article, we will know what is the Muslim population in Sri Lanka. Sri Lanka is an island country located in the northern part of the Indian Ocean in South Asia. The distance of this country located in the south of India is only 31 kilometers from India. Until 1972 its name was Ceylon, which was changed to Lanka in 1972 and Sri Lanka by adding the respectful word "Sri" in 1978. In 2022, the population of Sri Lanka is 2.20 crore.
What is the Muslim population in Sri Lanka
At present the Muslim population in Sri Lanka is around 21 lakhs, which is about 10% of the total population. Buddhism is the largest religion in Sri Lanka with about 70% of the population. Here people of Hindu religion come second. The Sri Lankan Muslim community is divided into Sri Lankan Moors, Indian Moors and Sri Lankan Malays based on their history and traditions.
Sri Lankan Moors are of diverse origin, with some tracing their ancestry to Arab traders who first settled in Sri Lanka around the 9th century, and who intermarried with local women. The concentration of Moors is highest in Ampara, Trincomalee and Batticaloa districts.
History of Muslims in Sri Lanka
By the 7th century, Arab traders controlled much of the trade on the Indian Ocean, including Sri Lanka. Many of these traders settled in Sri Lanka, encouraging the spread of Islam.
However, when the Portuguese arrived in Sri Lanka during the 16th century, many Muslim descendants of the Arabs were persecuted, thus forcing them to migrate to the Central Highlands and the East Coast.
In modern times, Muslims in Sri Lanka have the Department of Muslim Religious and Cultural Affairs, which was established in the 1980s to prevent the continued isolation of the Muslim community from the rest of Sri Lanka. Today, about 10% of Sri Lankans practice Islam, mostly from the Moor and Malay ethnic communities on the island.
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Online First articles are the published Version of Record, made available as soon as they are finalized and formatted. They are in general accessible to current subscribers, until they have been included in an issue, which is accessible to subscribers to the relevant volume
Continuity and change in the evolution of French yes-no questionsAuthor(s): Philip Comeau, Ruth King and Carmen L. LeBlancAvailable online: 13 June 2022More LessAbstract
This present study contributes to research on the structure of yes-no questions in French. Informed by previous historical linguistic research tracing developments from the Old French period onwards, we focus on qualitative analysis of grammatical commentary and variationist analysis of Acadian French spoken-language data. We compare the evolution of yes-no questions in Acadian, Metropolitan, and Quebec French, reconstructing the history of variants up to the present. While in most cases we encounter slow-moving change, we do find inter-varietal differences in degree of retention of individual variants, including outright loss; in development of stylistic differentiation; and in analogically based innovation. We also find inter-varietal differences in grammatical constraints governing usage and in the fine detail regarding sentential polarity, illuminated in terms of the semantico-pragmatic functions of negative yes-no questions. The overall results underline the importance of considering sociolinguistic histories, including histories of dialect contact, along with local linguistic markets.
Correlated grammaticalizationAuthor(s): David GoldsteinAvailable online: 01 June 2022More LessAbstract
Grammaticalization is characterized by robust directional asymmetries (e.g., Kuteva et al. 2019 ). For instance, body-part nominals develop into spatial adpositions, minimizers develop into negation markers and subject pronouns become agreement markers. Changes in the opposite direction are either rare or unattested ( Garrett 2012 : 52). Such robust cross-linguistic asymmetries have led some scholars to reify grammaticalization trajectories as universal mechanistic forces ( Heath 1998 : 729). One consequence of such a view is that the ambient morphosyntax of a language has little or even no relevance for grammaticalization. This paper uses Bayesian phylogenetic methods to demonstrate the critical role that pre-existing morphosyntax can play in grammaticalization. The empirical basis for this claim is the grammaticalization of definite and indefinite articles in the history of Indo-European: indefinite articles developed at a faster rate among languages in which a definite article had already emerged compared to those lacking a definite article. The two changes are thus correlated. The results of this case study suggest that there is much more to be learned about when and why grammaticalization occurs by investigating its relationship to the pre-existing linguistic system (cf. Reinöhl and Himmelmann 2017 : 381).
Origins of Dogon NP tonosyntaxAuthor(s): Jeffrey HeathAvailable online: 01 June 2022More LessAbstract
Like other complex morphosyntactic and morphophonological structures that are endemic to single language families, Dogon NP tonosyntax is the result of the fortuitous interlocking of diachronically unrelated processes and constructions. It arose due to the following combination: (a) right-headed prosodic pattern in noun-modifier sequences; (b) loss of numeral classifiers which had previously protected nouns from the right-headed prosodic pattern when followed by numerals; and (c) transfer of tones from possessors to following possessums. Although none of these phenomena were semantically driven, the resulting configuration could only be reinterpreted by native speakers in semantic terms, creating a completely new system unique to Dogon. In spite of having arisen accidentally, this tonosyntactic system is quite stable. One of its benefits is the unusual solution it provided (at no extra charge) to a perennial problem in the design of relative constructions.
Is Malayo-Polynesian a primary branch of Austronesian?Author(s): Victoria Chen, Jonathan Kuo, Maria Kristina S. Gallego and Isaac SteadAvailable online: 16 May 2022More LessAbstract
An understudied morphosyntactic innovation, reanalysis of the Proto-Austronesian (PAn) stative intransitive prefix *ma- as a transitive affix, offers new insights into Austronesian higher-order subgrouping. Malayo-Polynesian is currently considered a primary branch of Austronesian, with no identifiably closer relationship with any linguistic subgroup in the homeland ( Blust 1999 , 2009/2013 ; Ross 2005 ). However, the fact that it displays the same innovative use of ma- with Amis, Siraya, Kavalan and Basay-Trobiawan and shares the merger of PAn *C/t with this group suggests that Malayo-Polynesian and East Formosan may share a common origin – the subgroup that comprises the four languages noted above. This observation points to a revised subgrouping more consistent with a socio-historical picture where the out-of-Taiwan population descended from a seafaring community expanding to the Batanes and Luzon after having developed a seafaring tradition. It also aligns with recent findings in archaeology and genetics that (i) eastern Taiwan is the most likely starting point of Austronesian dispersal ( Hung 2005 , 2008 , 2019 ; Bellwood 2017 ; Bellwood & Dizon 2008 ; Carson & Hung 2018 ) and (ii) that the Amis bear a significantly closer relationship with Austronesian communities outside Taiwan ( Capelli et al. 2001 ; Trejaut et al. 2005 ; McColl et al. 2018 ; Pugach et al. 2021 ; Tätte et al. 2021 ). Future investigation of additional shared innovations between Malayo-Polynesian and East Formosan could shed further light on their interrelationships.
Copying form without contentAuthor(s): Brigitte PakendorfAvailable online: 26 April 2022More LessAbstract
Two major types of change are generally distinguished in language contact studies: the transfer of linguistic form (frequently taken to include transfer of concomitant meaning or function) and the transfer of structural and semantic patterns by themselves, without attendant form. A type of change that is less frequently discussed is so-called relexification. This involves the transfer of form without model-language semantic or syntactic specifications that is grafted onto an equivalent recipient-language lemma. Relexification has been suggested to play a role in the development of mixed languages or creoles, but as is shown here, it can also be identified in several ordinary situations of language contact from around the world. This type of change represents a mirror image of the transfer of patterns without lexical material and supports recent models of language selection in bilinguals.
Drastic demographic events triggered the Uralic spreadAvailable online: 08 April 2022More LessAbstract
The widespread Uralic family offers several advantages for tracing prehistory: a firm absolute chronological anchor point in an ancient contact episode with well-dated Indo-Iranian; other points of intersection or diagnostic non-intersection with early Indo-European (the Late Proto-Indo-European-speaking Yamnaya culture of the western steppe, the Afanasievo culture of the upper Yenisei, and the Fatyanovo culture of the middle Volga); lexical and morphological reconstruction sufficient to establish critical absences of sharings and contacts. We add information on climate, linguistic geography, typology, and cognate frequency distributions to reconstruct the Uralic origin and spread. We argue that the Uralic homeland was east of the Urals and initially out of contact with Indo-European. The spread was rapid and without widespread shared substratal effects. We reconstruct its cause as the interconnected reactions of early Uralic and Indo-European populations to a catastrophic climate change episode and interregionalization opportunities which advantaged riverine hunter-fishers over herders.
Preverbal a-marking in Palenquero CreoleAuthor(s): Hiram L. SmithAvailable online: 25 March 2022More LessAbstract
Formally similar grammatical features in a creole and its genetic or areal relatives may indicate substrate transfer, lexifier influence, or grammaticalization. Against this backdrop, the present study investigates the origin(s) of the preverbal past marker a in Palenquero Creole (Colombia). Results from distributional analysis and tests for significance indicate that several diachronically-related meanings are a-marked at rates approaching obligatory, suggesting advanced grammaticalization. Comparative results for Peninsular haber + PP suggest that past marking has grammaticalized much further in half the time in Palenquero Creole than in its lexifier, Spanish. Why? I argue, against traditional accounts about the origins of a, that, given the contact history of Palenquero speakers, most likely a pre-existing Kikongo prefixal form merged with an already grammaticalizing haber, thus propelling grammaticalization in the creole. The synchronic patterning shows adherence to typological patterns observed for perfectives in line with well-known constraints on competition and selection in contact languages, such as their grammatical congruence or particular social ecologies.


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|Reviews for Reparations
| Guest chapter 10 . 2/6/2019
You have such a great writing style and the pacing of your chapters were greatly, well, paced heh. Also I love how so well in tune Draco in your story is compared to the Draco in the books/movies. It was really fun to read and nice to see them in a healing setting (I got so many Grey's Anatomy vibes). I can't wait to read Foundations!
| Guest chapter 10 . 1/27/2019
I've loved every second of this story and I'm very excited to continue on 'Foundations'. Thank you for publishing it!
| Guest chapter 10 . 1/6/2019
Loved your story!
I think that the development of Draco's and Harry's relationship was really amazing, because while there were certainly important scenes I can't point my finger on what really made the difference. I felt that they stayed in character the whole time and it didn't feel rushed! Thank you so much for this :")
| BeautifullyTwistd chapter 10 . 10/20/2018
Wow. I absolutely loved this fic! Really really enjoyed it. Perfect length, perfect characterisation and perfect amount of plot going on. So good. Thank you.
| BeautifullyTwistd chapter 7 . 10/19/2018
This is so good!
| Silvanesti chapter 10 . 10/10/2018
A beautiful and highly entertaining story, thank you.
| CaliforniaCougar chapter 10 . 9/22/2018
Absolute perfection. I gotta say, I almost didn't read past the first chapter because apparently stories not in past tense irk my sensibilities, but your writing was too good to walk away from. A delightful read from start to finish.
| CaliforniaCougar chapter 2 . 9/20/2018
I love the hostile interactions between them already. hilarious.
| DaisyPotter13 chapter 10 . 9/18/2018
loved this!
| Guest chapter 9 . 8/14/2018
| Guest chapter 6 . 8/14/2018
Chapter 6 is my fav chapter yet! It made me smile and laugh and both harry and draco's characters seem really spot-on. Love this fic- putting it on the list of all time fav drarry! Great job 3
| bethabetha chapter 6 . 8/14/2018
it's so rare to find a ff in which the characters are *believable* and this is one of those rare stories. i'm a very picky reader, and i honestly can't find a single negative thing to say about Reparations. the canon characters are both original and true to their canon selves, and i really like the OCs. great premise, great plot so far, and great writing
| JanieOhio chapter 10 . 7/31/2018
OMG. I loved this so much. great job writing these two characters so well. rushing off to the sequel.
| Guest chapter 9 . 7/26/2018
| Guest chapter 9 . 7/26/2018
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MONTPELLIER, July 17, 1816.
SIR—Your favor of the 12th instant overrates the hospitality for which your visit afforded me the agreeable opportunity. If otherwise, it would have been overpaid by the sentiments which it has led you to express.
It is very gratifying to be assured from such a source, that not only your Monarch, inheriting the virtues of a Brother whose generous policy was allied to the early fortunes of our Country, but that the French nation, cherishes friendly dispositions and liberal views towards it. The United States are faithful to a system of justice, of good will, and of intercourse mutually beneficial with all nations; and they are sensible of all the considerations which strengthen these motives in relation to France. They take a sincere interest in her independence, her prosperity, and her happiness; and they cannot better express it than in their wishes that the French people may enjoy all the just advantages, external and internal, of a regular and free Constitution, adapted to their genius and satisfactory to their desires.
I hope, sir, that the partialities you indulge for our Country will find all the returns which are due to amiable qualities adorning distinguished nations.
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Chicago: James Madison Jr., "To M. Hyde De Neuville.," Writings of James Madison, Volume 3 in James Madison, Letters and Other Writings of James Madison, 4 Vols. (Philadelphia: J.B. Lippincott & Co.), P.14 Original Sources, accessed August 8, 2022, http://www.originalsources.com/Document.aspx?DocID=51BIKPV1UIPFSNB.
MLA: Madison, James, Jr. "To M. Hyde De Neuville." Writings of James Madison, Volume 3, in James Madison, Letters and Other Writings of James Madison, 4 Vols. (Philadelphia: J.B. Lippincott & Co.), P.14, Original Sources. 8 Aug. 2022. http://www.originalsources.com/Document.aspx?DocID=51BIKPV1UIPFSNB.
Harvard: Madison, J, 'To M. Hyde De Neuville.' in Writings of James Madison, Volume 3. cited in , James Madison, Letters and Other Writings of James Madison, 4 Vols. (Philadelphia: J.B. Lippincott & Co.), P.14. Original Sources, retrieved 8 August 2022, from http://www.originalsources.com/Document.aspx?DocID=51BIKPV1UIPFSNB.