Fig 2: REDD1 deficiency cooperates with KRAS to drive tumor progression and metastatic dissemination. (A) Kaplan-Meier analysis of p48-Cre;KrasG12D; Redd1−/− (p48KR) mice (n = 6, median survival = 364 d) and p48-Cre;KrasG12D;Redd1+/+ (p48K) mice (n = 12, median survival = 610.5 d). All animals euthanized exhibited PanIN and/or PDAC. P = 0.0005 by log-rank test. (B, top) representative gross photographs depicting enlarged, nodule-studded pancreas and enlarged spleen (left) and liver with metastatic foci (right) from 17-mo-old p48KR mice. (Bottom) Hematoxylin and eosin (H&E)-stained tissue sections from the indicated genotypes. The right panels represent higher magnification of the respective left panels. p48KR mice show PDAC and liver metastasis (indicated by an asterisk) compared with p48K mice that show early PanIN histology. Scale bars: left, 50 µm; right 20 µm. (C) Summary of histological findings of PDAC and metastasis including those reported in A and additional mice sacrificed at 11 mo. Total n = 20 mice analyzed per genotype. (D) Kaplan-Meier analysis of KrasG12D; Redd1+/+ and KrasG12D; Redd1−/−mice after intratracheal adenoviral Cre (AdK and AdKR mice, respectively). AdK mice (median survival = 229 d, n = 7); AdKR mice, median survival = 196 d, n = 13. Survival times are after infection. P = 0.011 by log-rank test. (E, left) Representative H&E-stained sections of whole-mount lung tissues from a pair of age- and sex-matched AdK and AdKR mice 20 wk after intratracheal delivery of Cre. Tumors are indicated by arrows. Scale bars, 2 mm. (Right) Quantification of lung tumor burden per lung lobe from mice of the denoted genotypes. Each dot indicates one lobe (n ≥ 2 mice for each time point per genotype). Horizontal lines indicate mean ± SEM. (*) P = 0.01, by two-tailed t-test. (F) Necropsy of AdKR mice euthanized 28 wk after intratracheal Cre treatment showing metastasis to kidney (K), diaphragm (D), and pancreas (P). H&E-stained sections of primary lung tumor and metastatic foci (indicated by an asterisk) are shown. Scale bars, 100 µm. See also Supplemental Figure S1.

Fig 3: Model for REDD1 loss in RAS-driven tumorigenesis. REDD1 loss up-regulates HIF, PPARγ/CD36, and fatty acid-binding proteins (FABPs) to mediate uptake and lipid droplet storage. This effect is associated with increased fatty acid oxidation (FAO) that generates ATP as well as reducing equivalents (NADPH and GSH) through the mitochondrial Krebs (TCA) cycle, resulting in relief from RAS-induced energy stress and oxidative stress, and ultimately tumor progression. We hypothesize that combination therapies targeting the multiple metabolic nodes described herein are likely to be effective against REDD1-deficient, RAS mutant cancers.

Fig 4: REDD1-dependent ROS controls autophagy and ATG4B activity.(a,b) Reduced H2O2 in Redd1−/− MEFs and splenocytes from Redd1−/− mice as compared with wild-type controls, assessed by staining with CM-H2DCFDA (3 μM) and then flow cytometry. Shown are the means from three independent experiments/mice measured in triplicate. (c) Tsc2−/− MEFs exhibit elevated cytosolic H2O2. (d) Decreased H2O2 induction in Redd1−/− versus wild-type cells on starvation (0.1% FBS). (e) Suppression of ATG4B activity by H2O2 (3 mM, 6 h) in Redd1−/− cells, assessed by incubation of cellular extracts with the LC3-PLA2 substrate at the indicated concentrations. Each data point for velocity is derived from 60 sequential measurements, with the mean and 95% confidence intervals (shown as error bars) calculated by regression analysis (see Methods). (f) H2O2 (3 mM, 6 h) restores LC3B processing in Redd1−/− cells to wild-type levels in the presence of CQ (30 μM, 4 h). Note similar levels of ATG4B in Redd1−/− cells and wild-type cells in the absence or presence of H202. β-Tubulin (Tub.), loading control. (g) Elevated ATG4B activity in Redd1−/− versus wild-type cells under basal conditions, assessed as described for e. (h) Persistent increase in ATG4B activity in Redd1−/− versus wild-type cells under starvation (0.1% FBS, 24 h). (i) Enhanced cleavage of unprocessed LC3, as evidenced by a decreased GFP/DsRed ratio (the mean fluorescence of expressing cells) in Redd1−/− compared with wild-type cells, transfected with double-tagged DsRed-LC3-GFP and analysed using flow cytometry. H2O2 (1 mM, 6 h) inhibits ATG4B to increase the GFP/DsRed ratio in Redd1−/− cells. (j) The mean and s.d. of four independent experiments as in i, measured in triplicate. ***P<0.001, **P<0.01, *P<0.05, by paired t-test (a–e) or repeated measures ANOVA (g,h).

Fig 5: REDD1 mediates autophagy independent of mTORC1.(a) Impaired starvation-induced autophagy in Redd1−/− cells despite suppressed mTORC1, assessed by the analysis of phosphorylated p70 S6 Kinase (pS6K, T389). Cells were cultured with Krebs media and treated in the absence or presence of CQ (30 μM, 1 h). At right, densitometry showing the mean values of three independent experiments and statistical comparison with respective wild-type lanes. (b) Rapamycin (100 nM, 4 h) fails to fully restore the defective autophagic flux in Redd1−/− cells, as assessed by LC3B processing in the absence or presence of CQ (30 μM, 4 h; compare lanes 4 and 8). Activity of mTORC1 was assessed via phosphorylated ribosomal protein S6 (pS6, S235/S236). Right, densitometry quantitation of three independent experiments as in a. (c) Failure of hypoxia (1% O2, 12 h) to induce autophagy in Redd1−/− cells (left) but not Tsc2−/− cells (right), assessed by degradation of GFP–LC3 measured using flow cytometry. (d) Both wild-type REDD1 and the mTORC1-inactive mutant REDD1-RPAA14 are sufficient to induce autophagy, assessed by GFP-LC3 levels using flow cytometry. Tetracycline (Tet)-inducible (TREX) cells were treated in the presence and absence of rapamycin (100 nM, 4 h) as a positive control. For c,d, shown are the means of two independent experiments, each performed as triplicate cultures; error bars, s.d. (*) show P-values compared with untreated control; (+) compares REDD1 with REDD1-RPAA. For all experiments: ***P<0.001, **P<0.01, *P<0.05; +P<0.05, by paired t-test.