Autophagy Sustains Pancreatic Cancer Growth through Both Cell-Autonomous and Nonautonomous Mechanisms

Abstract

Autophagy has been shown to be elevated in pancreatic ductal adenocarcinoma (PDAC), and its role in promoting established tumor growth has made it a promising therapeutic target. However, due to limitations of prior mouse models as well as the lack of potent and selective autophagy inhibitors, the ability to fully assess the mechanistic basis of how autophagy supports pancreatic cancer has been limited. To test the feasibility of treating PDAC using autophagy inhibition and further our understanding of the mechanisms of protumor effects of autophagy, we developed a mouse model that allowed the acute and reversible inhibition of autophagy. We observed that autophagy inhibition causes significant tumor regression in an autochthonous mouse model of PDAC. A detailed analysis of these effects indicated that the tumor regression was likely multifactorial, involving both tumor cell-intrinsic and host effects. Thus, our study supports that autophagy inhibition in PDAC may have future utility in the treatment of pancreatic cancer and illustrates the importance of assessing complex biological processes in relevant autochthonous models.Significance: This work demonstrates that autophagy is critical pancreatic tumor maintenance through tumor cell-intrinsic and -extrinsic mechanisms. These results have direct clinical relevance to ongoing clinical trials as well as drug-development initiatives. Cancer Discov; 8(3); 276-87. ©2018 AACR.See related commentary by Noguera-Ortega and Amaravadi, p. 266This article is highlighted in the In This Issue feature, p. 253.

Figure 1

Generation and functional analysis of the inducible Atg4B^CA mouse model. A, Tissue-specific promoter driven Cre expression will remove STOP cassette to express reverse tetracycline-controlled transactivator (rtTA). Doxycycline binds rtTA to turn on Tet-On cassette and induce Atg4B^C74A expression. B, Immunofluorescence staining of LC3 (green) and RFP (red) in Atg4B^CA+ tumor cells treated with or without Dox for 6 days. noD indicates no Dox treatment. Blue, DAPI stained nuclei. Scale bars: 5μM. C, Representative immunofluorescence staining of LC3 and RFP in Atg4B^CA++ tumor cells showing level of autophagy. Scale bars: 5μM. D, Quantification of LC3+ puncta per cell in Atg4B^CA+ and Atg4B^CA++ tumor cells. Dox treatment reduced autophagy level in basal (Top panel) or Chloroquine treated (+CQ at Bottom panel) condition indicating inhibited autophagic flux. More than 10 fields per cell line were measured. SD plotted as Error bars. **p<0.001, *p<0.05 by T-test. E, Western blot showing expression of RFP, LC3I/II in Atg4B^CA+ and Atg4B^CA++ cells treated with Dox for 3 days (3d) and 6 days (6d). F, Representative IHC images of PDACs stained for RFP with no expression (top panel), one copy (bottom left) and two copies (bottom right) expression of Atg4B^CA. Scale bars: 500μM. G, Representative IHC images of PDACs stained for LC3 with no (top panel), one copy (bottom left) and two copies (bottom right) of Atg4B^CA expression. Inserts were 3 folds enlargement of the white framed areas; autophagosomes (LC3+ puncta) were marked by white arrowhead. Scale bars: 10μM. H, Electronic microscope pictures showing ultrastructure of tumors from Atg4B− and Atg4B^CA++ tumors. Two samples of each genotype were examined and a total of 10 fields were measured in the graph. ***p<0.001 by T-test.

Figure 2

Impact of autophagy inhibition on PDAC growth in Atg4B^CA+ and Atg4B^CA++ mice. A, Comparison of tumor growth between Atg4B− control and Atg4B^CA+ mice. Tumor volumes were measured by ultrasound and relative growth was plotted against time (weeks) since initial tumor identification (0wk), Dox diet (625 mg/kg Dox) was given after tumor identification. Numbers of mice in each group at each time-point are indicated in the graph. B, Survival analysis of mice plotted in A. p-value by Log-rank test. C, Comparison of tumor growth among Atg4B− control, Atg4B^CA++ and Atg4B^CA++ intermittently induced (Int) groups. Tumor volumes were measured by ultrasound and relative growth was plotted against time (weeks) from initial tumor identification (0wk). Numbers of mice in each group at each time-point are indicated in the graph. D, Survival analysis of mice plotted in C. p-value by Log-rank test. E, Relative growth comparison between Atg4B− (n=28) and Atg4B^CA++ (n=21) tumors after Dox treatment for one week. **P<0.001 by T-test. F, Western blot of RFP, cl-Casp3 and LC3I/II in starved (HBSS treated) Atg4B− and Atg4B^CA++ tumor cells treated with or without Dox. G, IHC analysis of cl-Casp-3 staining in Atg4B− and Atg4B^CA+ tumors. Scale bar: 100μM. cl-Casp3+ cells/field was compared as shown in the right plot. Each dot represents one mouse with more than 5 fields measured per mouse. H, IHC analysis of Ki67 staining in Atg4B− and Atg4B^CA+ tumors. Scale bar: 50 μM. Proliferation index as Ki67+cell ratio was compared as shown in the right plot. Each dot represents one mouse with more than 5 fields measured per mouse.

Figure 3

The cell intrinsic and extrinsic effect of autophagy inhibition on tumor growth. A, Plot of colony number (mean+SD) formed in Atg4B− and Atg4B^CA++ cells. Each group contained 3 individual cell lines and each cell line was repeated three times. *P<0.05 by T-test. B, Plot of colony number and size formed in Atg4B− and Atg4B^CA++ cells grown in soft agar. Each group contained 3 individual cell lines and each cell line was repeated three times. *P<0.05 by T-test. C, Relative growth of Atg4B^CA++ and Atg4B− sub-cutaneous xenografts on Dox diet. n=10 in each group, *p<0.05. D, Relative growth of orthotopic pancreatic tumors. ***p<0.0001, *p<0.05. E, Representative images of CD68 IHC in Atg4B− and Atg4B^CA++ autochthonous tumors showing macrophage infiltration. Scale bar: 100 μM. Quantification of CD68+ cells per field showed significantly increased number of macrophages in the Atg4B^CA++ group (n=6) vs. Atg4B− group (n=6), 5–10 randomly selected fields were quantified for each tumor. **p=0.0021 by T-test. F, Representative images of CD68 IHC in Atg4B^CA++ tumors orthotopically injected in nude mice either on or off doxycycline food. Scale bar: 100 μM. There was no significant difference in macrophage infiltration between the Atg4B^CA++ (n=5) group and the Atg4B− group (n=5), 8 fields were randomly selected for each tumor. G, Relative growth of Atg4B^CA++ tumors treated with empty liposome (PBS-Lipo) (n=6) or clodronate liposome (Clod-Lipo, n=5). ***p<0.001, *p<0.05 by T-test.

Figure 4

Autophagy inhibition impairs tumor seeding efficiency through cell autonomous and non-cell autonomous mechanisms. A, Schematic experimental design to demonstrate how tumor take was affected by autophagy inhibition. B, Detection of tumor take with ultrasound after orthotopic injection of Atg4B^CA++ cells into Atg4B^CA++, Rosa-rtTA^LSL, Ubc-ERT/Cre⁺ mice on normal diet (BD− TU−, n=13)), Atg4B−,Ubc− mice on Dox diet (BD− TU++, n=11) and Atg4B^CA++, Rosa-rtTA^LSL, Ubc-ERT/Cre⁺ mice on Dox diet (BD++ TU++, n=16). Numbers of mice with detected tumors are shown in the graph. *p<0.05 by Fisher’s exact test. C, Relative tumor growth of orthotopically-injected tumors plotted in B. *p<0.05. D, Schematic of experimental design to assess the non-cell autonomous effect of autophagy inhibition on tumor take. E, Detection of tumor take with ultrasound after orthotopic injection of Atg4B − cells into Atg4B^CA++, Rosa-rtTA^LSL, Ubc-ERT/Cre⁺ mice on normal diet (BD− TU−, n=15) and on Dox diet (BD++ TU−, n=16). Numbers of mice with positive seeding were shown in the graph. **p<0.001, *p<0.05 by Fisher’s exact test F, Relative tumor growth of orthotopically-injected tumors plotted in C. No significant differences are seen between groups at any time points. G, Model of cell autonomous and non-cell autonomous mechanisms of autophagy inhibition impacting PDAC growth.