Rottlerin inhibits mTOR activation and autophagy in PaSC by AMPK-independent mechanisms.

<p>(A) Primary mouse PaSC were treated with 1 μM rottlerin and Western blotting analysis was used to determine the activation state of AMPK and the mTORC1 targets p70 S6K and 4E-BP1. (B and C) Immortalized mouse PaSC were transfected with non-targeting (“con”) or AMPKα1/2 siRNA (“AMPK”) and then treated with rottlerin at the indicated concentrations for 1 h or 24 h. (B) Levels of total and phosphorylated AMPK, p70 S6K, S6 and ERK (loading control) were measured by Western blotting. (C) Levels of the autophagic markers LC3, p62, and GAPDH (loading control) were measured by Western blotting. (D) Cellular metabolic state of mock-transfected or AMPKα1/2 siRNA transfected imPaSC treated with vehicle or rottlerin for 72 hours was assessed by MTT assay. Graph shows O.D. values relative to control cells (mean ±SEM). As indicated in the graph, the reduction in cellular metabolic state induced by rottlerin was comparable in mock- and siRNA transfected cells. Data is representative of 3 independent experiments; ns = no statistical significant differences between control and AMPK siRNA transfected cells (t-test).</p

Rottlerin induces autophagy dysregulation in PaSC.

<p>(A and B) Cells were treated with vehicle (-) or rottlerin for up to 48 h. Cellular protein levels of LC3-I and its LC3-phosphatidylethanolamine conjugate (LC3-II) were analyzed by Western blotting after short (A) and long-term treatment (B). Immunoblot in panel B also shows protein levels of the autophagy regulators p62/SQSTM1, LAMP-2, and Beclin-1. Fibronectin and α-SMA levels are shown as representative markers of stellate cell activation and loading controls. (C) Live imaging of GFP-LC3 mPaSC treated with vehicle or 2.5 μM rottlerin for up to 24 h by fluorescence microscopy. Representative pictures show diffuse cytoplasmic distribution of GFP-LC3 in control cells and GFP-LC3 puncta formation upon treatment with rottlerin. (D) mPaSC were immunostained for p62/SQSTM1 (green staining) and DAPI was used for visualization of nuclei. Pictures show progressive formation of p62 aggregates in mPaSC upon rottlerin treatment. (E and F) Blockade of autophagy by lysosomal inhibitors does not affect LC3 lipidation in mPaSC. Cells were pre-incubated for 30 min with bafilomycin A1 (E) or a lysosomal inhibitor cocktail (bafilomycin A1 + pepstatin A + E-64; panel F), and then incubated for 1 or 24 h with 1 μM rottlerin. Protein levels of LC3 and α-SMA were measured by Western blotting as autophagy marker and loading controls.</p

Rottlerin induces rapid mitochondrial dysfunction in mPaSC that precedes LC3 puncta formation.

<p>(A) mPaSC isolated from GFP-LC3 transgenic mice were labeled with 25 nM MitoTracker (MITO; red fluorescence) for 10 min and then treated without (control) or with 2.5 μM rottlerin for the indicated times. MITO staining (red) and GFP-LC3 puncta (green fluorescence) formation in live cells were visualized using a fluorescence microscope. (B) Rate of cellular oxygen consumption measured in live imPaSC under basal conditions and after sequential additions of DMSO and 1 μM rottlerin. Data is expressed as fold change relative to basal. Graph shows mean ± SEM; 3 independent experiments; * p<0.05 as compared to basal (t-test). (C) mPaSC were treated with 1 μM rottlerin for 30 or 60 minutes and total cellular ATP levels were measured in cell lysates by luminescence assay. Graph shows mean ± SEM for 3 independent experiments; * p<0.05 as compared to control (t-test). (D) mPaSC treated with 2.5 μM rottlerin for 1 or 24 hours were double immunostained for the mitochondrial marker Tom20 (green staining) and the stellate cell marker α-SMA (red staining); nuclei were counter-stained with DAPI (blue staining). Images were visualized under fluorescence microscope.</p

CHOP regulates cell death in rottlerin-treated PaSC.

<p>(A and B) mPaSC were treated with different concentrations of rottlerin at the indicated times. Activation of the PERK/eIF2α branch of the UPR measured by the protein levels of total and phosphorylated eIF2α and the proapoptotic transcription factor CHOP. GAPDH expression was analyzed as a loading control. As shown, CHOP upregulation could be detected as early as 15 min in cells treated with 1 μM rottlerin (A) and this effect was sustained for at least 24 h (B). (C) Immunofluorescence for CHOP (red staining) in nuclei (blue DAPI staining) of mPaSC treated with 1μM rottlerin for 3 h and 24 h. (D) AMPKα1/2 were silenced by siRNA in imPaSC and then cells treated with rottlerin at indicated concentrations for 1 h or 24 h. p-eIF2α, total eIF2α, CHOP and GAPDH (loading control) were measured by Western blotting. (E) mPaSC isolated from wild type (WT) or <i>Chop -/-</i> mice were treated with rottlerin for 24 h. Immunoblots show protein levels of CHOP, GRP78, LC3 and GAPDH (loading control). (F and G) Cell death was assessed in WT or <i>Chop -/-</i> mPaSC treated with rottlerin for 48 h. Apoptosis was determined by DNA fragmentation ELISA (panel F) and cell number (panel G). Data in graphs is presented as mean ±SEM, n = 3; * p<0.05 as compared to WT; # p<0.05 as compared to control at 0 μM rottlerin (two way ANOVA followed by post-hoc Tukey tests). (H and I) p62 (panel H) and death receptor 5 (Dr5; panel I) mRNA levels were determined by qPCR in rottlerin-treated WT and <i>Chop -/-</i> mPaSC. As indicated, rottlerin-induced upregulation of p62 and Dr5 were blunted in <i>Chop -/-</i> cells. Data in graphs is presented as mean ±SEM, n = 3–4; * p<0.05 as compared to WT; # p<0.05 as compared to time 0 (two way ANOVA followed by post-hoc Tukey tests).</p

Incidence of pancreatic cancer is dramatically increased by a high fat, high calorie diet in KrasG12D mice

<div><p>Epidemiologic data has linked obesity to a higher risk of pancreatic cancer, but the underlying mechanisms are poorly understood. To allow for detailed mechanistic studies in a relevant model mimicking diet-induced obesity and pancreatic cancer, a high-fat, high-calorie diet (HFCD) was given to <i>P48</i>^<i>+/Cre</i><i>;LSL-KRAS</i>^<i>G12D</i> (KC) mice carrying a pancreas-specific oncogenic Kras mutation. The mice were randomly allocated to a HFCD or control diet (CD). Cohorts were sacrificed at 3, 6, and 9 months and tissues were harvested for further analysis. Compared to CD-fed mice, HFCD-fed animals gained significantly more weight. Importantly, the cancer incidence was remarkably increased in HFCD-fed KC mice, particularly in male KC mice. In addition, KC mice fed the HFCD showed more extensive inflammation and fibrosis, and more advanced PanIN lesions in the pancreas, compared to age-matched CD-fed animals. Interestingly, we found that the HFCD reduced autophagic flux in PanIN lesions in KC mice. Further, exome sequencing of isolated murine PanIN lesions identified numerous genetic variants unique to the HFCD. These data underscore the role of sustained inflammation and dysregulated autophagy in diet-induced pancreatic cancer development and suggest that diet-induced genetic alterations may contribute to this process. Our findings provide a better understanding of the mechanisms underlying the obesity-cancer link in males and females, and will facilitate the development of interventions targeting obesity-associated pancreatic cancer.</p></div

HFCD promotes inflammation in the pancreas of KC mice.

<p>Inflammatory parameters in the histological sections of pancreas were evaluated quantitatively. Specifically, acinar loss scores, inflammatory scores, fibrosis scores, and pancreatitis indices were determined for male and female KC mice fed the CD or HFCD for different time periods. The values are means ± SD. *<i>P</i><0.05, Student’s <i>t</i>-tests.</p

HFCD leads to greater weight gain in male and female KC mice.

<p><b>(A)</b> A schematic view of the study design. <b>(B)</b> Weight gain of male (left panel) and female (right panel) KC mice fed the CD or HFCD. The values are means ± SD. *<i>P</i><0.05, Student’s <i>t</i>-tests. For the male mice collected at 3, 6, and 9 months, n = 12 (5 on CD and 7 on HFCD), 12 (6 on CD and 6 on HFCD), and 11 (5 on CD and 6 on HFCD), respectively. For the female mice collected at 3, 6, and 9 months, n = 11 (6 on CD and 5 on HFCD), 12 (5 on CD and 7 on HFCD), and 11 (5 on CD and 6 on HFCD), respectively. <b>(C)</b> Plasma levels of insulin, leptin, cholesterol, glucose, and triglycerides in 9-month-old KC mice fed the CD or HFCD. Data are depicted as means ± SD. *<i>P</i><0.05 vs. control, Student’s <i>t</i>-tests.</p

HFCD markedly accelerates stroma formation, extracellular matrix deposition and exocrine atrophy in KC mice.

<p><b>(A)</b> The extent of pancreatic collagen deposition was evaluated by Sirius red staining. Graph shows percentage of Sirius red-stained area in pancreas tissue sections at the indicated ages. Data represent mean ± SEM; 8–10 random pancreatic sections were evaluated per mouse; 3–4 mice per group. *<i>P</i><0.05 vs. CD. <b>(B)</b> Pictures illustrate Sirius red staining in pancreatic tissue sections of KC mice fed the CD or HFCD for 6 months, the time-point displaying the highest differences in collagen deposition between CD-fed and HFCD-fed mice. <b>(C)</b> Pancreatic levels of fibrosis-related proteins were analyzed by Western blotting in pancreas lysates from KC mice fed the CD or HFCD for 9 months. Picture shows representative immunoblots of fibronectin; prolyl-4-hydroxylase (P4HA2), a key collagen processing enzyme; cadherin 11, a mesenchymal marker expressed by activated myofibroblasts; α-SMA, a myofibroblast marker; and p-STAT3 (Y705)/ total STAT3. Picture also shows protein levels of pancreatic amylase, a digestive enzyme produced by acinar cells and GAPDH used as loading control. Each lane represents an individual mouse; three mice per group are shown. <b>(D)</b> Graphs show optical density of immunoblots depicted in panel D. Data in graphs represent mean ± SEM, n = 3. *<i>P</i><0.05 vs. CD.</p

HFCD leads to an accumulation of autophagic vacuoles and p62/SQSTM1.

<p><b>(A,D,F)</b> Representative IF images of LC3-II <b>(A,F)</b>, p62/SQSTM1 (p62) <b>(D,F)</b> in PanIN lesions <b>(A,D)</b>, and histologically normal exocrine pancreas <b>(F)</b> of KC mice of indicated age fed CD and high fat calorie diet HFCD. DAPI was used to stain nuclei, and DIC to visualize tissue structure (upper lines). Scale bar: 20 μm. <b>(B)</b> Immunoblot analysis of LC3 in pancreas from 3- and 9-month-old KC mice. GAPDH is a loading control. <b>(C,E,G)</b> Quantification of LC3-II <b>(C,G)</b> and p62 <b>(E,G)</b> integral intensity, average size of puncta and % positively stained area was performed with ImageJ software. The values are means ± SEM (3 mice were analyzed for each strain, age and diet). * <i>P</i>< 0.05 vs. 3 months (mo) CD, # <i>p</i> < 0.0001 vs. 3 mo HFCD, $ <i>p</i> < 0.05 vs. 9 mo CD, ƒ <i>p</i>< 0.05 vs. 3 months (mo) CD.</p

Histological analysis of the pancreas.

<p>Pancreas histology of male and female KC mice fed the CD or HFCD for 3, 6, and 9 months. For each group, images (10x) from two different mice were shown. CA, cancer.</p