The HMGB1/RAGE inflammatory pathway promotes pancreatic tumor growth by regulating mitochondrial bioenergetics - PubMed (original) (raw)
The HMGB1/RAGE inflammatory pathway promotes pancreatic tumor growth by regulating mitochondrial bioenergetics
R Kang et al. Oncogene. 2014.
Abstract
Tumor cells require increased adenosine triphosphate (ATP) to support anabolism and proliferation. The precise mechanisms regulating this process in tumor cells are unknown. Here, we show that the receptor for advanced glycation endproducts (RAGE) and one of its primary ligands, high-mobility group box 1 (HMGB1), are required for optimal mitochondrial function within tumors. We found that RAGE is present in the mitochondria of cultured tumor cells as well as primary tumors. RAGE and HMGB1 coordinately enhanced tumor cell mitochondrial complex I activity, ATP production, tumor cell proliferation and migration. Lack of RAGE or inhibition of HMGB1 release diminished ATP production and slowed tumor growth in vitro and in vivo. These findings link, for the first time, the HMGB1-RAGE pathway with changes in bioenergetics. Moreover, our observations provide a novel mechanism within the tumor microenvironment by which necrosis and inflammation promote tumor progression.
Conflict of interest statement
CONFLICT OF INTEREST
The authors declare no conflict of interest.
Figures
Figure 1
HMGB1 promotes ATP production and subsequent cell growth, cell migration and NF-κB activation in pancreatic tumor cells. (a) Time course and dose-response effects of HMGB1 on ATP production and cell growth in cultured pancreatic tumor cell lines. The relative ATP level, with untreated control set as 1 (mean±s.d., n = 3), is shown. (b) Panc02 cells were treated with HMGB1 (10µg/ml) in the presence or absence of trichostatin A (400 n
m
), and then assayed for ATP levels and cellular proliferation. Data represent relative levels, with untreated control (ctrl) set as 1 (mean±s.d., n = 3, **P<0.001, NS = not significant). (c) Lysed (necrotic) MEFs lacking HMGB1 do not elicit production of ATP at 24 h in Panc02 tumor cells. Data shown are the relative ATP levels, with the untreated ctrl set as 1 (mean±s.d., n = 3, **P<0.001). (d) Panc02 cells were treated with lysed 3 × 105 necrotic (‘Nec’) or apoptotic (‘Apo’) MEFs for 24 h in the presence or absence of HMGB1 Ab (50 µg/ml) or control IgG (left panel), and then assayed for ATP levels (right panel). Panc02 cells were treated with HMGB1 (10 µg/ml) for 24 h in the presence or absence of Dnase I (10 U/ml) and antibodies (50 µg/ml) as indicated, and then assayed for ATP levels (right panel). Relative ATP levels, with untreated ctrl set as 1 (mean±s.d., n = 3, **P<0.001), are shown. (e) Pancreatic tumor cells were treated with HMGB1 (10 µg/ml) in the presence or absence of Rote (0.5, 1 and 2 µ
m
), and then assayed for ATP levels, NF-κB activity by luciferase reporter assay, migration by transwell assay, and cell growth by counting cell number. Data represent relative level, with untreated ctrl set as 1 (mean±s.d., n = 3, *P<0.01, **P<0.001). (f) After transfection with p65 shRNA or ctrl shRNA for 48h, Panc02 cells were treated with HMGB1 (10 µg/ml) for 24 h and the ATP level was then analyzed. Data represent relative levels, with untreated ctrl shRNA set as 1 (mean±s.d., NS = not significant).
Figure 2
RAGE is required for HMGB1-mediated ATP production. (a) Pancreatic tumor cells were transfected with specific shRNA (top panel) for 48 h or treated with the indicated antibody (Ab, 10 µg/ml, bottom panel) for 12 h, then treated with HMGB1 (10 µg/ml) for 24 h and assayed for ATP production. Data represent relative ATP levels, with untreated control set as 1 (mean±s.d., _n_= 3, *P <0.05, **P <0.001, NS: not significant). (b) RAGE knockdown inhibits mitochondrial complex I activity with or without HMGB1 (10 µg/ml) treatment for 24 h. Data represent relative levels of ATP and complex I activity, with untreated control shRNA set as 1 (mean±s.d., n =3, **P <0.001). (c, d) RAGE knockdown (c) or wild-type, (d) Panc02 cells were transfected with ‘cyt-RAGE’ or full RAGE (‘full-RAGE’) as indicated, treated with or without U0126 (10 µ
m
), Bay 11–7085 (10 µ
m
), rapamycin (100 n
m
) and indomethacin (300 µ
m
) for 1 h, and then treated with HMGB1 (10 µg/ml) for 24 h. Data represent relative levels of ATP and cellular proliferation, with untreated control set as 1 (mean±s.d., n = 3, **P <0.001).
Figure 3
HMGB1 treatment is associated with enhanced expression of RAGE in mitochondria. (a) Cytoplasmic (Cyto), and mitochondrial (Mit) was extracted from pancreatic tumor cells and pancreatic tissues were separated by SDS–PAGE. (b) Increasing amounts of mitochondrial extracts as indicated were probed for RAGE, tubulin and mitHSP70. (c) Mitochondria were isolated from pancreatic tumor cells and treated with 100 ng/ml of proteinase K for 15min in the absence or the presence of 1% Triton X-100. Samples were probed for RAGE, cytochrome oxidase IV, GRIM-19 and Bcl-2 by western blot. (d) Panc02 cells were treated with 10 µg/ml HMGB1 for 24 h, and antibodies to complex I–IV (CxI–IV) or a nonspecific control IgG were incubated with Panc02 mitochondrial extracts. Immunoprecipitates were resolved on SDS–PAGE and probed for RAGE, CxI NDUFA9, CxII Fp subunit, CxIII core protein 2 and CxIV subunit I by western blot. (e) RAGE knockdown (‘RAGE shRNA’) and wild-type (‘ctrl shRNA’) Panc02 cells were pretreated with 5µg/ml HMGB1 for 24 h, and then treated with oligomycin (1 u
m
), p-trifluoromethoxy carbonyl cyanide phenyl hydrazone (‘FCCP’, 0.3 u
m
), ‘2DG’, 100 m
m
, and Rote, 1 u
m
sequentially as indicated. OCR and ECR were monitored using the Seahorse Bioscience Extracellular Flux Analyzer in real time (mean±s.d., n =3). (f) Western blot analysis indicated protein in samples from whole or mitochondrial extract from pancreatic tumor (‘T’) or nearby normal control (‘N’) tissues. Representative western blots (top panel) and quantifications of five patient tissues (bottom) are shown. (g) Panc02 cells were treated with 5–10 µg/ml HMGB1 for 24 h. Western blot analysis was then performed to detect RAGE, actin or mitHSP70 in whole cells or mitochondrial extracts. Relative mitRAGE (bottom panel) is shown as mean±s.d. (_n_=3 *P <0.001 versus control).
Figure 4
Ser377 is required for RAGE activity in mitochondria. (a) Panc02 cells were treated with 10 µg/ml HMGB1 for 24 h in the absence or the presence of the ERK inhibitors U0126 and PD98059 (20 µ
m
), followed by western blot analysis of whole cells or mitochondrial extracts. Quantitative data is shown in the right panel, with normal ctrl set as 1 or 0.1 (mean±s.d., n = 3, *P <0.001 versus single HMGB1 group). (b) Knockdown of MEK2 by siRNA in Panc02 cells decreased relative levels of p-RAGE, p-CxI, mitRAGE, and ATP with or without 10 µg/ml HMGB1 treatment for 24 h. Data is shown as mean±s.d., with the normal control set as 1 or 0.1 (n = 3, *P <0.001 versus ctrl siRNA group). (c) uantitative analysis of p-ERK in wild-type and RAGE knockdown Panc02 cells following treatment with a representative damage-associated molecular pattern molecule (HMGB1), pathogen-associated molecular pattern molecule (lipopolysaccharide), cytokine (TNFα) or chemotherapeutic drug (melphalan, oxaliplatin) as indicated for 24h (mean±s.d., _n_=3, *P <0.001 versus ctrl shRNA group). (d) Antibodies to RAGE, p-ERK1/2 or a nonspecific control IgG were incubated with Panc02 extracts following 10 µg/ml HMGB1 treatment with or without U0126 (20 µm) for 12 h. Immunoprecipitates were resolved on SDS–PAGE and probed for RAGE, p-ERK1/2. (e) Panc02 cells were transfected with full-RAGE, ex-RAGE, m-RAGE and cyt-RAGE plasmids fused with a GFP-tag. Antibodies to GFP, p-ERK1/2 or a nonspecific control IgG were incubated with Panc02 extracts as indicated. Immunoprecipitates were resolved on SDS–PAGE and probed for GFP, p-ERK1/2. (f) Panc02 cells were treated with 10 µg/ml HMGB1 for 24 h, followed by western blot analysis of cytosolic (‘Cyt’) or mitochondrial (‘Mit’) extracts as indicated. (G) Panc02 cells 10 µg/ml HMGB1 for 24 h in the absence or the presence of cycloheximide (CHX, 10 µg/ml), followed by western blot analysis of mitochondrial extracts. (h) Panc02 cells were transfected with full-RAGE and exm-RAGE plasmids fused with a GFP-tag, then treated with 10 µg/ml HMGB1 for 24 h. western blot analysis of membrane (‘Mem’) and mitochondrial (‘Mit’) extracts. (i) Panc02 cells were transfected with full-RAGE (‘WT’) and mutants as indicated, and treated with 10 µg/ml HMGB1 for 24 h. Relative levels of mitRAGE, p-CxI, and ATP are shown as mean±s.d., with normal ctrl set as 1 or 0.1 (n =3, *P <0.001 versus normal WT group, #P <0.01, NS: not significant).
Figure 5
RAGE is required for HMGB1-mediated ATP production. (a) C57/BLl6 mice were inoculated with 106 Panc02 tumor cells following transfection of control or RAGE-specific shRNA and treated with an HMGB1 inhibitor, ‘EP’, 40 mg/kg, or PBS beginning on day 7. Tumors were measured twice weekly, and volumes were calculated for 42 days (mean ±s.d., n = 5, *P<0.001 ctrl shRNA versus RAGE shRNA; #P<0.01 ctrl shRNA versus ctrl shRNA + EP). (b) On day 42 in the experiments described in (a), serum HMGB1 (left) and protein levels of C-PARP, Bcl-2, p-p65 and LC3-I/II (right) in tumor samples were assayed by western blotting. The relative optical intensity of the HMGB1-immunoreactive band (in arbitrary units, AU) is shown in a scatter plot with the mean represented by a solid line (n = 5, *P<0.001 versus ctrl shRNA). The inset shows a representative western blot. In parallel, relative ATP levels and complex I activity in tumor tissue were evaluated, with ctrl shRNA set as 1 (mean ± s.d., n = 3, *P <0.001 versus ctrl shRNA). (c) Dose-response effects of EP on cellular proliferation in Panc02 and Panc2.03 cells. (d) On day 42 as described in (a), the percentage of CD11b positive cells in tumor tissue was evaluated (mean ± s.d., _n_=3, *P<0.001). (e) RAGE wild-type (+/+) and knockout (−/−) mice were inoculated with 0.5–1 × 106 Panc02 tumor cells. Tumors were measured twice weekly, and tumor volumes calculated for 42 days (mean ± s.d., _n_=5, *P<0.001 +/+ versus −/−). (f) On day 42 described in (e), ATP levels in tumor tissue (1 × 106) were determined, with normal RAGE wild-type (+/+) set as 1 (mean±s.d., n = 3, *P<0.001 versus +/ +).
Figure 6
Model of HMGB1/RAGE interaction and regulation of metabolism. HMGB1 is released following stress/necrosis in cells as a damage-associated molecular pattern molecule in the tumor microenvironment, promoting inflammation. HMGB1 binds cell membrane RAGE or uptake into cells by a caveolin-dependent endocytosis pathway, promoting phosphorylation of ERK1/2 (p-ERK1/2), which in turn induces phosphorylation of RAGE. Phosphorylation of RAGE at Ser377 is required for its mitochondria-targeted accumulation. There it promotes phosphorylation of complex I, which is the first step in the ATP biosynthetic pathway. As shown in the summary, the HMGB1–RAGE pathway stimulates bioenergetics by enhancing ATP production in a p-ERK1/2-dependent process to sustain the increased metabolic needs and growth of tumor cells.
References
- Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144:646–674. - PubMed
- Vakkila J, Lotze MT. Inflammation and necrosis promote tumour growth. Nat Rev Immunol. 2004;4:641–648. - PubMed
- Lotze MT, Tracey KJ. High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal. Nat Rev Immunol. 2005;5:331–342. - PubMed
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