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p53 status determines the role of autophagy in pancreatic tumour development

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Abstract

Macroautophagy (hereafter referred to as autophagy) is a process in which organelles termed autophagosomes deliver cytoplasmic constituents to lysosomes for degradation1. Autophagy has a major role in cellular homeostasis and has been implicated in various forms of human disease2,3,4. The role of autophagy in cancer seems to be complex, with reports indicating both pro-tumorigenic and tumour-suppressive roles3,5,6,7,8,9,10,11,12. Here we show, in a humanized genetically-modified mouse model of pancreatic ductal adenocarcinoma (PDAC), that autophagy’s role in tumour development is intrinsically connected to the status of the tumour suppressor p53. Mice with pancreases containing an activated oncogenic allele of Kras (also called Ki-Ras)—the most common mutational event in PDAC13—develop a small number of pre-cancerous lesions that stochastically develop into PDAC over time. However, mice also lacking the essential autophagy genes Atg5 or Atg7 accumulate low-grade, pre-malignant pancreatic intraepithelial neoplasia lesions, but progression to high-grade pancreatic intraepithelial neoplasias and PDAC is blocked. In marked contrast, in mice containing oncogenic Kras and lacking p53, loss of autophagy no longer blocks tumour progression, but actually accelerates tumour onset, with metabolic analysis revealing enhanced glucose uptake and enrichment of anabolic pathways, which can fuel tumour growth. These findings provide considerable insight into the role of autophagy in cancer and have important implications for autophagy inhibition in cancer therapy. In this regard, we also show that treatment of mice with the autophagy inhibitor hydroxychloroquine, which is currently being used in several clinical trials14, significantly accelerates tumour formation in mice containing oncogenic Kras but lacking p53.

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Figure 1: Loss of autophagy in the pancreas causes organ destruction and reduces survival.
Figure 2: Autophagy-deficient mice show enhanced Ras-driven PanIN formation but do not develop PDAC.
Figure 3: Genetic or pharmacological inhibition of autophagy in the absence of p53 permits and accelerates PDAC formation.
Figure 4: Loss of p53 results in lower levels of autophagy, and combined loss of p53 and Atg7 enhances levels of glycolytic and pentose phosphate pathway intermediates.

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Acknowledgements

We are grateful to M. Komatsu for providing the Atg7 floxed mice that were used in this study, and we thank M. O’Prey for help with fluorescent microscopy; G. Gillen for help with PET; T. McBryan for help with statistics; C. Frezza for help with metabolic studies; and A. Rosenwald for help and advice on pathology. This work was supported by Cancer Research UK and the Pancreatic Cancer Research Fund.

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Authors and Affiliations

Authors

Contributions

M.T.R. and K.M.R. conceived the study. M.T.R., E.G., P.D.A., K.I.A., O.J.S. and K.M.R. designed experiments. M.T.R., J.O.P., J.P.M., C.N., G.M., A.M., A.A., T.S.R., L.Z. and R.R. conducted experiments. M.T.R., J.P.M., A.R., E.G., P.D.A., K.I.A., O.J.S. and K.M.R. analysed experiments. M.T.R. and K.M.R. wrote the manuscript. J.O.P. and J.P.M. contributed equally to this study.

Corresponding author

Correspondence to Kevin M. Ryan.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Effects of loss of autophagy on pancreatic tissue and pancreatic function.

a, Representative examples of pancreatic acinar tissue stained with haematoxylin and eosin, p53 and caspase-3 from mice of the indicated genotypes. Mice were killed at specified time points. In wild-type acini (Kras+/+Atg7+/+) p53 and caspase-3 staining is virtually completely negative at all time points. In Atg7−/− acinar tissue, p53 levels and caspase-3 activation are markedly enhanced at 35 days of age. At later time points p53 levels and caspase-3 activation are maintained, albeit at a lower level (quantified in Fig. 1c). Scale bars: 50 μm. b, Representative examples of pancreatic endocrine tissue stained with haematoxylin and eosin, p53 and caspase-3 from mice of the indicated genotypes that have been killed at defined time points. For better identification, islets are encircled in most panels. Wild-type islets (Pdx Atg7+/+) are histologically normal at all time points; p53 and caspase-3 staining is virtually completely absent. Haematoxylin and eosin staining of islets in Atg7−/− islets (Pdx Atg7−/−) shows progressive morphological disintegration over time. At 35 days of age, islets look histologically largely normal but cells balloon and progressively disintegrate over time. This destruction is not accompanied by significant p53 accumulation or caspase-3 activation in islets at all times. Notably all increases in p53 levels and caspase-3 activation are confined to acinar tissue. Scale bars: 50 μm. c, Biochemical analysis of pancreatic function. Endocrine function was assessed by measuring blood serum levels of glucose and fructosamine. Exocrine function was assessed by a combination of parameters: blood cholesterol, blood triglycerides, blood amylase, blood lipase and faecal elastase. These were not altered in moribund Pdx-Cre Atg7−/− mice compared to age-matched wild-type mice. Detailed information about both groups is provided in the table. Values are expressed as median ± s.d. A Mann–Whitney U-test was used to ascertain significance.

Extended Data Figure 2 Loss of Atg5 in the pancreas mimics the phenotype of Atg7 loss.

a, Serial sections of the same pancreatic region stained with haematoxylin and eosin, LC3, p62, p53 and caspase-3 showing Atg5−/− next to Atg5+/+ areas after Pdx-Cre-mediated mosaic recombination in a 101-day-old mouse. Recombined tissue (−/−) is in the lower right corner and Atg5 wild-type tissue in the upper left corner of the panels. Representative regions (+/+ and −/−) are cropped (from the area identified by the rectangle) and provided underneath the main panels. Like their Atg7−/− counterparts, Atg5−/− areas show strong diffuse staining for LC3, p62 aggregates, and have increased levels of p53 and caspase-3 activation. Non-recombined areas have a distinct, punctate LC3 staining pattern, do not accumulate p62, do not elevate p53, and do not activate caspase-3. b, Kaplan–Meier analysis comparing overall survival of mice either Atg5+/+(blue) or Atg5−/− (red) in the pancreas. Note that the Atg5+/+ cohort is the same cohort as the Atg7+/+ cohort in Fig. 1d (blue). Median survival ± s.d. and male/female (m/f) are provided in the table. A log-rank test (Mantel–Cox) was used for statistical analysis. n/a#, 14/16 animals were culled >500 days when completely healthy. As in Pdx-Cre Atg7−/− mice, loss of Atg5 reduces survival in a large proportion of mice. c, Representative haematoxylin and eosin, p53 and caspase-3 images of Atg5-deficient exocrine and endocrine (islets) pancreatic tissue from mice with an average age of 113 days show increased p53 and caspase-3 activity only in acinar tissue but not in islets. Notably islets display the same destructive morphological changes as Atg7−/− islets. d, Assessment of endocrine (glucose, fructosamine) and exocrine blood serum parameters shows a diabetic phenotype (increased glucose and fructosamine) in moribund Pdx-Cre Atg5−/− mice but not their age-matched Atg5+/+ counterparts. Exocrine pancreatic blood parameters are not altered in moribund Atg5−/− mice. Faecal elastase levels as a read-out for pancreatic exocrine function was also not different between both groups. Detailed information about both groups is provided. Values are expressed as median ± s.d. For statistical analysis a Mann–Whitney U-test was used. ***P < 0.001. Scale bars: 50 μm.

Extended Data Figure 3 Additional deletion of p53−/− in Pdx-Cre Atg7−/− mice delays early death.

a, Kaplan–Meier analysis comparing overall survival of Pdx-Cre Atg7−/− mice that are either p53+/+ (red) or p53−/− (black). Median survival ± s.d. and male/female (m/f) are provided in the table. Additional deletion of p53 in Atg7−/− pancreases only delays the early death but does not rescue mortality rate. A log-rank test (Mantel–Cox) was used for statistical analysis. b, Representative haematoxylin and eosin, caspase-3 images from Atg7-deficient exocrine and endocrine tissue that is either p53+/+ (left two columns) or p53−/− (right two columns). For each genotype and staining a lower magnification (×20) and a higher (×40) magnification panel is shown containing both exocrine (acini) and endocrine (islets) tissue. Acinar caspase-3 levels are significantly reduced in p53−/− Atg7−/− pancreases compared to p53+/+ Atg7−/− pancreases. Islets are caspase-3 negative and show similar destructive morphological changes in both groups. c, Caspase-3 activation is quantified (median, s.e.m.) and statistical analysis was done using a Mann–Whitney U-test. The male/female ratio is provided. d, Assessment of endocrine (glucose, fructosamine) and exocrine blood serum parameters shows a diabetic phenotype (increased glucose and fructosamine) in moribund Pdx-Cre p53−/− Atg7−/−mice but not their age-matched Pdx-Cre p53−/− Atg7+/+ counterparts. Exocrine blood parameters and faecal elastase levels (another read-out for pancreatic exocrine function) are not different between both groups. Values are expressed as median ± s.d. For statistical analysis, a Mann–Whitney U-test was used. **P < 0.01, *P < 0.05. Detailed information about the mice is provided. e, Kaplan–Meier analysis comparing overall survival of mice that are either p53-proficient (blue) or p53-deficient (green) in the pancreas. Atg7 is wild type in both cohorts. n/a#, no death was observed for 200 days and mice were killed in complete health. f, Haematoxylin and eosin staining from pancreatic tissue of p53+/+ and p53−/− mice (93 days of age) is provided in a lower overview magnification (×20, left column) and a higher magnification (×40, right column). Morphology is not altered in p53−/− pancreases. Scale bars: 100 μm (lower magnification panels), 50 μm (higher magnification).

Extended Data Figure 4 Changes in pancreases from Pdx-Cre KrasG12D/+ Atg7+/+ and Pdx-Cre KrasG12D/+ Atg7−/− mice over time.

a, Alcian blue/PAS (AB/PAS), Atg7, LC3 and p62 staining in pancreases from 150-day-old Atg7-proficient and Atg7-deficient KrasG12D/+ mice. b, Representative pancreatic sections from Pdx-Cre KrasG12D/+ Atg7+/+ mice stained with haematoxylin and eosin, p53 and caspase-3 from mice killed at the indicated time points are shown. Magnified cropped regions are identified by a dotted rectangle. PanINs at 35 days are rare but increase in number at later time points (compare Fig. 2b). PanINs are always AB/PAS positive and have elevated p53 and p21 levels. Caspase-3 activation is very rare at all time points. Scale bars: 100 μm. c, Representative pancreatic sections from Pdx-Cre KrasG12D/+ Atg7−/− mice stained with haematoxylin and eosin, p53 and caspase-3 from mice killed at indicated time points are shown. Magnified cropped regions are identified by a dotted rectangle. PanINs at 35 days are rare but rapidly increase in number at later time points. PanINs are always AB/PAS positive and have increased p53 (compare Fig. 2g) and p21 levels. Caspase-3 activation is very pronounced at later time points (compare Fig. 2g). Scale bars: 100 μm.

Extended Data Figure 5 Analysis of p53 and caspase-3 activation, cell proliferation and biochemical analysis of endocrine and exocrine pancreatic function in Pdx-Cre KrasG12D/+ Atg7+/+ versus Pdx-Cre KrasG12D/+ Atg7−/− mice.

a, Assessment of endocrine (glucose, fructosamine) and exocrine blood serum parameters shows a diabetic phenotype (increased glucose and fructosamine) in moribund Pdx-Cre KrasG12D/+ Atg7−/− mice but not in their Pdx-Cre KrasG12D/+ Atg7+/+counterparts. Exocrine pancreatic blood parameters are not altered in ill mice. Faecal elastase levels as a read-out for pancreatic exocrine function are not different between both groups. Values are expressed as median ± s.d. b, Caspase-3 (casp3), p21 and SaβGal stainings from 150-day-old mice of the indicated genotypes. c, p53 and caspase-3 activation of the indicated genotypes were quantified (median, s.e.m.) in mice of 90 and 150 days of age. The male/female ratio is provided. d, Representative examples of PanINs stained with Ki67 from mice of the indicated genotypes killed at defined time points. Scale bars: 50 μm. e, Quantification of percentage of Ki67 positive cells in PanINs from mice of the indicated genotypes killed at defined time points. Five PanINs from five mice (90 d Atg7+/+, 90 d Atg7−/−, 150 d Atg7+/+) and five PanINs from four mice (150 d Atg7−/−) were scored. For statistical analysis a Mann–Whitney U-test was used.

Extended Data Figure 6 Loss of Atg5 in the pancreas of Pdx-Cre KrasG12D/+ mice mimics the phenotype of Atg7 loss in pancreases expressing mutant KrasG12D.

Representative sections stained with haematoxylin and eosin, AB/PAS, LC3, p62, p53 and caspase-3 of pancreatic tissue from on average 93-day-old mice. PanIN number is increased compared to autophagy-proficient pancreases expressing mutant KrasG12D (overview haematoxylin and eosin and AB/PAS panels). PanINs stain positive for Alcian blue/PAS, p53 and p21. Loss of autophagy is shown by p62 accumulation and a diffuse LC3 staining pattern. Islets in the overview panels are encircled. Scale bars: 200 μm (dotted), 50 μm (solid).

Extended Data Figure 7 Tumour-bearing Pdx-Cre KrasG12D/+ p53−/− Atg7+/+ and Pdx-Cre KrasG12D/+ p53−/− Atg7−/− mice show no differences in exocrine and endocrine pancreatic function and tumours from Pdx KrasG12D/+ p53−/− Atg5−/− mice are autophagy-deficient.

All mice for this figure were moribund and had to be killed because of malignancy. a, Assessment of endocrine (glucose, fructosamine) and exocrine blood serum parameters does not show a difference between both genotypes. Likewise, faecal elastase activity is not different between both groups. Values are expressed as median ± s.d. b, Haematoxylin and eosin, LC3 and p62 staining of a tumour from a Pdx-Cre KrasG12D/+ p53−/− Atg5−/− mouse confirms that the tumour is arising from autophagy-deficient tissue as evidenced by strong, diffuse LC3 staining and p62 accumulation. For each staining a representative overview and a magnified cropped region to show LC3 and p62 staining specifics are identified by a dotted rectangle. Scale bars: 100 μm.

Extended Data Figure 8 Effects of chloroquine treatment on the pancreas of Pdx-Cre KrasG12D/+ p53+/+ Atg7+/+ mice and on pancreatic tumours from Pdx-Cre KrasG12D/+ p53−/− Atg7+/+ mice.

a, Histology of tumours from Pdx-Cre KrasG12D/+ p53−/− Atg7+/+ mice treated with chloroquine. Shown are haematoxylin and eosin and LC3 stains of representative tumours from Pdx-Cre KrasG12D/+ p53−/− Atg7+/+ mice treated with vehicle control (PBS) or chloroquine (CQ). Chloroquine-treated mice show stronger LC3 staining indicating inhibition of autophagy. Scale bars: 100 μm. b, Representative haematoxylin and eosin images of pancreases from Pdx-Cre KrasG12D/+ treated with vehicle control (PBS) or chloroquine from 51 to 59 days of age for 36 days. For each group an overview panel and a representative higher magnification panel of exocrine pancreatic tissue (acini), endocrine tissue (islets) and a PanIN is provided. Scale bars: 200 μm (dotted), 50 μm (solid). There are no evident histological differences between both groups. c, PanIN quantification. Chloroquine treatment does not alter the number of PanINs (Mann–Whitney U-test). d, Assessment of endocrine (glucose, fructosamine) and exocrine blood serum parameters does not show a difference between both genotypes. Likewise, faecal elastase activity is not different between both groups. Values are expressed as median ± s.d. A Mann–Whitney U-test test was used to ascertain significance.

Extended Data Figure 9 Atg7 deficiency affects autophagic flux, but does not alter intracellular TCA cycle intermediates, acetyl-CoA levels in cells or PanIN number in tissue from Pdx KrasG12D/+ p53−/− mice nor does it significantly affect steady-state levels of glycolytic, TCA or pentose phosphate pathway intermediates in pre-cancerous PDEC cultures.

a, Quantification of LC3-II levels in the western blot shown in Fig. 4b. A Mann–Whitney U-test was used to ascertain significance. b, Western blot and densitometry analysis of LC3-II accumulation in p53-proficient and p53-deficient cell lines after treatment with lysosomal protease inhibitors leupeptin/NH4Cl indicating greater autophagic flux in the former. Quantification is provided (densitometry of LC3-II/p38). Values are the average of four biological replicates for each genotype and treatment. Error bars are s.e.m. Statistical analysis was via a t-test. c, Intracellular TCA-cycle metabolites were assessed by LC-MS and expressed as fold difference compared to Atg7+/+ tumour cell lines (Pdx KrasG12D/+ p53−/−). Values represent the average of eight (Atg7+/+) and six (Atg7−/−) biological replicates. Error bars are s.e.m. Differences are not significant (NS, Mann–Whitney U-test). d, Atg7 deletion (red, n = 5) does not increase the number of PanIN per mm2 tissue in mice killed 21 days after birth compared to Atg7 proficient mice of the same age (blue, n = 3). Detailed information about mice used for the analysis is provided. e, Western blot analysis of ductal epithelial cells isolated from four different KrasG12D-driven p53+/+ pancreases that are either Atg7-proficient or Atg7-deficient. Lack of Atg7 immunoreactivity and lack of LC3-I to LC3-II conversion in Atg7−/− cultures confirm autophagy deficiency. f, LC-MS of intracellular glycolytic and pentose phosphate pathway intermediates shows no statistically significant difference between both genotypes. g, Intracellular TCA-cycle metabolites show no statistically significant difference between both genotypes. Values in f and g represent the average of three biological replicates. Errors bars are s.e.m. Differences are not significant (NS, Mann–Whitney U-test).

Extended Data Figure 10 Re-expression of Atg7 in Atg7−/− tumour cell lines rescues autophagy but does not revert the metabolic profile.

Murine Atg7 or empty control vector (pBabe) was retrovirally infected to seven different Pdx-Cre KrasG12D/+ p53−/−Atg7−/− cell lines. a, Representative immunofluorescence images of Pdx KrasG12D/+ p53−/−Atg7−/− cells that are left uninfected, express empty vector (pBabe) or mAtg7 show that Atg7 is expressed in virtually all cells infected with pBabe mAtg7. b, Western blot analysis shows strong Atg7 expression after retroviral expression and re-established LC3-I to LC3-II conversion. c, Autophagic flux in Atg7 reconstituted cells was assessed with treatment of lysosomal protease inhibitors leupeptin/NH4Cl for 2 h. In all cell lines treatment with leupeptin/NH4Cl increased LC3-II levels, indicating that autophagic flux is fully restored. d, e, Extracellular acidification rate (ECAR, d) and average oxygen consumption rate (OCR, e) of seven different Atg7−/− cell lines (blue) is not affected by retroviral infection of either empty vector pBabe (red) or Atg7 (green). Values are the average of seven biological replicates for each group. Error bars are s.e.m.

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Rosenfeldt, M., O’Prey, J., Morton, J. et al. p53 status determines the role of autophagy in pancreatic tumour development. Nature 504, 296–300 (2013). https://doi.org/10.1038/nature12865

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