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Neutrophil elastase-mediated degradation of IRS-1 accelerates lung tumor growth.

Houghton AM, Rzymkiewicz DM, Ji H, Gregory AD, Egea EE, Metz HE, Stolz DB, Land SR, Marconcini LA, Kliment CR, Jenkins KM, Beaulieu KA, Mouded M, Frank SJ, Wong KK, Shapiro SD - Nat. Med. (2010)

Bottom Line: Recent data suggest that tumor-associated inflammatory cells may modify lung tumor growth and invasiveness.This study identifies IRS-1 as a key regulator of PI3K within malignant cells.Additionally, to our knowledge, this is the first description of a secreted proteinase gaining access to the inside of a cell and altering intracellular signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

ABSTRACT
Lung cancer is the leading cause of cancer death worldwide. Recent data suggest that tumor-associated inflammatory cells may modify lung tumor growth and invasiveness. To determine the role of neutrophil elastase (encoded by Elane) on tumor progression, we used the loxP-Stop-loxP K-ras(G12D) (LSL-K-ras) model of mouse lung adenocarcinoma to generate LSL-K-ras-Elane(-/-) mice. Tumor burden was markedly reduced in LSL-K-ras-Elane(-/-) mice at all time points after induction of mutant K-ras expression. Kaplan-Meier survival analysis showed that whereas all LSL-K-ras-Elane(+/+) mice died, none of the mice lacking neutrophil elastase died. Neutrophil elastase directly induced tumor cell proliferation in both human and mouse lung adenocarcinomas by gaining access to an endosomal compartment within tumor cells, where it degraded insulin receptor substrate-1 (IRS-1). Immunoprecipitation studies showed that, as neutrophil elastase degraded IRS-1, there was increased interaction between phosphatidylinositol 3-kinase (PI3K) and the potent mitogen platelet-derived growth factor receptor (PDGFR), thereby skewing the PI3K axis toward tumor cell proliferation. The inverse relationship identified between neutrophil elastase and IRS-1 in LSL-K-ras mice was also identified in human lung adenocarcinomas, thus translating these findings to human disease. This study identifies IRS-1 as a key regulator of PI3K within malignant cells. Additionally, to our knowledge, this is the first description of a secreted proteinase gaining access to the inside of a cell and altering intracellular signaling.

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Neutrophil Elastase induces tumor cell proliferation(a) 3H uptake for LSL-K-ras tumor-derived cell lines5 co-incubated with WT and Elane−/− PMN for two hours. Results from a representative experiment in triplicate. Bars ± SEM. *P<0.05 from control. **P=0.011, ANOVA. (b) 3H uptake for LSL-K-ras cells stimulated with NE or vehicle for 60 min. Data from a representative experiment in triplicate. Bars ± SEM. *P<0.01 from NE=0. (c) 3H uptake and (d) cell counts for A549 cells and 3H uptake for (e) 201T cells (K-ras WT). Results from representative experiments in triplicate. Bars ± SEM, P<0.05 from NE=0. (f) 3H uptake for A549 cells stimulated with NE in presence or absence of 1.0 μM LY294002 or 10 μM U0126 for 60 min. Results from a representative experiment in triplicate. Bars +/− SEM. P<0.05. (g) Western blots of pAkt, Akt and B-actin for NE-exposed lysates of A549, 201T, and LSL-K-ras cells. (h) Western blot of phospho-p44/42-MAPK, p44-42-MAPK, pEGFR, and B-actin for NE-exposed A549 lysates. (i) Confocal images for EEA1, caveolin-1, and NE from A549 cells exposed to AlexaFluor488-conjugated NE or vehicle. Nuclei were counterstained using DAPI. (j) 3H uptake for A549 cells stimulated with NE ± 40 μM dynasore. Results from a representative experiment in triplicate. Bars +/− SEM. *P<0.05.
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Figure 2: Neutrophil Elastase induces tumor cell proliferation(a) 3H uptake for LSL-K-ras tumor-derived cell lines5 co-incubated with WT and Elane−/− PMN for two hours. Results from a representative experiment in triplicate. Bars ± SEM. *P<0.05 from control. **P=0.011, ANOVA. (b) 3H uptake for LSL-K-ras cells stimulated with NE or vehicle for 60 min. Data from a representative experiment in triplicate. Bars ± SEM. *P<0.01 from NE=0. (c) 3H uptake and (d) cell counts for A549 cells and 3H uptake for (e) 201T cells (K-ras WT). Results from representative experiments in triplicate. Bars ± SEM, P<0.05 from NE=0. (f) 3H uptake for A549 cells stimulated with NE in presence or absence of 1.0 μM LY294002 or 10 μM U0126 for 60 min. Results from a representative experiment in triplicate. Bars +/− SEM. P<0.05. (g) Western blots of pAkt, Akt and B-actin for NE-exposed lysates of A549, 201T, and LSL-K-ras cells. (h) Western blot of phospho-p44/42-MAPK, p44-42-MAPK, pEGFR, and B-actin for NE-exposed A549 lysates. (i) Confocal images for EEA1, caveolin-1, and NE from A549 cells exposed to AlexaFluor488-conjugated NE or vehicle. Nuclei were counterstained using DAPI. (j) 3H uptake for A549 cells stimulated with NE ± 40 μM dynasore. Results from a representative experiment in triplicate. Bars +/− SEM. *P<0.05.

Mentions: We examined the possibility that NE could directly induce tumor cell proliferation and performed co-culture experiments utilizing WT and Elane−/− PMN to demonstrate an essential requirement for NE in PMN-mediated tumor cell proliferation (Fig. 2a). Neutrophils only release ~2% of their NE content upon activation resulting in modest concentrations (~50 nM) just beyond the cell surface16. Dose response curves in LSL-K-ras tumor-derived cell lines (Fig. 2b) confirmed that modest concentrations of NE (40-80 nM) induced cellular proliferation, while excessive concentrations caused cell death (Fig. 2b). We reproduced NE-induced proliferation in two human lung adenocarcinoma cell lines, A549 (K-ras mutant) and 201T (K-ras WT)(Fig. 2c–e). The effects of NE required catalytic activity, as inactive NE (heated or synthetic inhibitor) failed to induce proliferation (Supplementary Fig. 2).


Neutrophil elastase-mediated degradation of IRS-1 accelerates lung tumor growth.

Houghton AM, Rzymkiewicz DM, Ji H, Gregory AD, Egea EE, Metz HE, Stolz DB, Land SR, Marconcini LA, Kliment CR, Jenkins KM, Beaulieu KA, Mouded M, Frank SJ, Wong KK, Shapiro SD - Nat. Med. (2010)

Neutrophil Elastase induces tumor cell proliferation(a) 3H uptake for LSL-K-ras tumor-derived cell lines5 co-incubated with WT and Elane−/− PMN for two hours. Results from a representative experiment in triplicate. Bars ± SEM. *P<0.05 from control. **P=0.011, ANOVA. (b) 3H uptake for LSL-K-ras cells stimulated with NE or vehicle for 60 min. Data from a representative experiment in triplicate. Bars ± SEM. *P<0.01 from NE=0. (c) 3H uptake and (d) cell counts for A549 cells and 3H uptake for (e) 201T cells (K-ras WT). Results from representative experiments in triplicate. Bars ± SEM, P<0.05 from NE=0. (f) 3H uptake for A549 cells stimulated with NE in presence or absence of 1.0 μM LY294002 or 10 μM U0126 for 60 min. Results from a representative experiment in triplicate. Bars +/− SEM. P<0.05. (g) Western blots of pAkt, Akt and B-actin for NE-exposed lysates of A549, 201T, and LSL-K-ras cells. (h) Western blot of phospho-p44/42-MAPK, p44-42-MAPK, pEGFR, and B-actin for NE-exposed A549 lysates. (i) Confocal images for EEA1, caveolin-1, and NE from A549 cells exposed to AlexaFluor488-conjugated NE or vehicle. Nuclei were counterstained using DAPI. (j) 3H uptake for A549 cells stimulated with NE ± 40 μM dynasore. Results from a representative experiment in triplicate. Bars +/− SEM. *P<0.05.
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Figure 2: Neutrophil Elastase induces tumor cell proliferation(a) 3H uptake for LSL-K-ras tumor-derived cell lines5 co-incubated with WT and Elane−/− PMN for two hours. Results from a representative experiment in triplicate. Bars ± SEM. *P<0.05 from control. **P=0.011, ANOVA. (b) 3H uptake for LSL-K-ras cells stimulated with NE or vehicle for 60 min. Data from a representative experiment in triplicate. Bars ± SEM. *P<0.01 from NE=0. (c) 3H uptake and (d) cell counts for A549 cells and 3H uptake for (e) 201T cells (K-ras WT). Results from representative experiments in triplicate. Bars ± SEM, P<0.05 from NE=0. (f) 3H uptake for A549 cells stimulated with NE in presence or absence of 1.0 μM LY294002 or 10 μM U0126 for 60 min. Results from a representative experiment in triplicate. Bars +/− SEM. P<0.05. (g) Western blots of pAkt, Akt and B-actin for NE-exposed lysates of A549, 201T, and LSL-K-ras cells. (h) Western blot of phospho-p44/42-MAPK, p44-42-MAPK, pEGFR, and B-actin for NE-exposed A549 lysates. (i) Confocal images for EEA1, caveolin-1, and NE from A549 cells exposed to AlexaFluor488-conjugated NE or vehicle. Nuclei were counterstained using DAPI. (j) 3H uptake for A549 cells stimulated with NE ± 40 μM dynasore. Results from a representative experiment in triplicate. Bars +/− SEM. *P<0.05.
Mentions: We examined the possibility that NE could directly induce tumor cell proliferation and performed co-culture experiments utilizing WT and Elane−/− PMN to demonstrate an essential requirement for NE in PMN-mediated tumor cell proliferation (Fig. 2a). Neutrophils only release ~2% of their NE content upon activation resulting in modest concentrations (~50 nM) just beyond the cell surface16. Dose response curves in LSL-K-ras tumor-derived cell lines (Fig. 2b) confirmed that modest concentrations of NE (40-80 nM) induced cellular proliferation, while excessive concentrations caused cell death (Fig. 2b). We reproduced NE-induced proliferation in two human lung adenocarcinoma cell lines, A549 (K-ras mutant) and 201T (K-ras WT)(Fig. 2c–e). The effects of NE required catalytic activity, as inactive NE (heated or synthetic inhibitor) failed to induce proliferation (Supplementary Fig. 2).

Bottom Line: Recent data suggest that tumor-associated inflammatory cells may modify lung tumor growth and invasiveness.This study identifies IRS-1 as a key regulator of PI3K within malignant cells.Additionally, to our knowledge, this is the first description of a secreted proteinase gaining access to the inside of a cell and altering intracellular signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

ABSTRACT
Lung cancer is the leading cause of cancer death worldwide. Recent data suggest that tumor-associated inflammatory cells may modify lung tumor growth and invasiveness. To determine the role of neutrophil elastase (encoded by Elane) on tumor progression, we used the loxP-Stop-loxP K-ras(G12D) (LSL-K-ras) model of mouse lung adenocarcinoma to generate LSL-K-ras-Elane(-/-) mice. Tumor burden was markedly reduced in LSL-K-ras-Elane(-/-) mice at all time points after induction of mutant K-ras expression. Kaplan-Meier survival analysis showed that whereas all LSL-K-ras-Elane(+/+) mice died, none of the mice lacking neutrophil elastase died. Neutrophil elastase directly induced tumor cell proliferation in both human and mouse lung adenocarcinomas by gaining access to an endosomal compartment within tumor cells, where it degraded insulin receptor substrate-1 (IRS-1). Immunoprecipitation studies showed that, as neutrophil elastase degraded IRS-1, there was increased interaction between phosphatidylinositol 3-kinase (PI3K) and the potent mitogen platelet-derived growth factor receptor (PDGFR), thereby skewing the PI3K axis toward tumor cell proliferation. The inverse relationship identified between neutrophil elastase and IRS-1 in LSL-K-ras mice was also identified in human lung adenocarcinomas, thus translating these findings to human disease. This study identifies IRS-1 as a key regulator of PI3K within malignant cells. Additionally, to our knowledge, this is the first description of a secreted proteinase gaining access to the inside of a cell and altering intracellular signaling.

Show MeSH
Related in: MedlinePlus