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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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NE promotes lung tumor growth in vivo(a) Kaplan-Meier Survival curve for AdenoCre recipient LSL/K-ras/Elane+/+ and LSL/K-ras/Elane−/− mice; P=0.006, log-rank test. (b) Tumor area (%) for both groups at 8, 14, and 20 weeks post-AdenoCre. N=5 mice per group. Bars ± SEM. *P<0.01. (c) BALF neutrophil counts for AdenoCre recipient LSL/K-ras/Elane+/+ and LSL/K-ras/Elane−/− mice at the 14-week time point. N=5 mice per group. Bars ± SEM. P=NS. Representative H&E images at 14 weeks (d, e) and 20 weeks (f) post-AdenoCre. (g) Representative images for anti-p40phox (anti-neutrophil) IHC at 14 weeks post-AdenoCre. (h) Representative Ki-67 IHC 14 weeks post-AdenoCre. (i) Ki-67 (+) cells per tumor area for N=5 mice per group at the 14-week time point. Bars ± SEM. *P<0.01. (j) Representative IF images and quantification for pAkt and phospho-MEK/ERK from both groups of mice 14 weeks post-AdenoCre. Bars ± SEM. P<0.01 for pAkt.
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Figure 1: NE promotes lung tumor growth in vivo(a) Kaplan-Meier Survival curve for AdenoCre recipient LSL/K-ras/Elane+/+ and LSL/K-ras/Elane−/− mice; P=0.006, log-rank test. (b) Tumor area (%) for both groups at 8, 14, and 20 weeks post-AdenoCre. N=5 mice per group. Bars ± SEM. *P<0.01. (c) BALF neutrophil counts for AdenoCre recipient LSL/K-ras/Elane+/+ and LSL/K-ras/Elane−/− mice at the 14-week time point. N=5 mice per group. Bars ± SEM. P=NS. Representative H&E images at 14 weeks (d, e) and 20 weeks (f) post-AdenoCre. (g) Representative images for anti-p40phox (anti-neutrophil) IHC at 14 weeks post-AdenoCre. (h) Representative Ki-67 IHC 14 weeks post-AdenoCre. (i) Ki-67 (+) cells per tumor area for N=5 mice per group at the 14-week time point. Bars ± SEM. *P<0.01. (j) Representative IF images and quantification for pAkt and phospho-MEK/ERK from both groups of mice 14 weeks post-AdenoCre. Bars ± SEM. P<0.01 for pAkt.

Mentions: We subjected Lox—Stop—Lox K-rasG12D/Elane−/− (LSL-K-ras/Elane−/−) and control (LSL-K-ras/Elane+/+) mice to 5×106 pfu intratracheal adenoviral cre recombinase (AdenoCre) to activate mutant K-ras expression4. During the 28 weeks following AdenoCre administration, all LSL-K-ras/Elane+/+ but none of the LSL-K-ras/Elane−/− mice died. Survival analysis demonstrated a significant (P=0.006) advantage for LSL-K-ras/Elane−/− mice (Fig. 1a). NE-deficiency is not completely protective, as we have subsequently identified death beyond 30 weeks in independent studies. Tumor burden was markedly reduced in LSL-K-ras/Elane−/− mice at all time points (Fig. 1b,d–f). The differences observed represent a reduction in tumor growth and differentiation (less mature lesions), as tumor number was equivalent in the two groups (Supplementary Table 1). NE-mediated effects on tumor growth are not model specific, as similar reductions in tumor growth were observed in the Lewis Lung carcinoma model using WT and Elane−/− mice (Supplementary Fig. 1).


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)

NE promotes lung tumor growth in vivo(a) Kaplan-Meier Survival curve for AdenoCre recipient LSL/K-ras/Elane+/+ and LSL/K-ras/Elane−/− mice; P=0.006, log-rank test. (b) Tumor area (%) for both groups at 8, 14, and 20 weeks post-AdenoCre. N=5 mice per group. Bars ± SEM. *P<0.01. (c) BALF neutrophil counts for AdenoCre recipient LSL/K-ras/Elane+/+ and LSL/K-ras/Elane−/− mice at the 14-week time point. N=5 mice per group. Bars ± SEM. P=NS. Representative H&E images at 14 weeks (d, e) and 20 weeks (f) post-AdenoCre. (g) Representative images for anti-p40phox (anti-neutrophil) IHC at 14 weeks post-AdenoCre. (h) Representative Ki-67 IHC 14 weeks post-AdenoCre. (i) Ki-67 (+) cells per tumor area for N=5 mice per group at the 14-week time point. Bars ± SEM. *P<0.01. (j) Representative IF images and quantification for pAkt and phospho-MEK/ERK from both groups of mice 14 weeks post-AdenoCre. Bars ± SEM. P<0.01 for pAkt.
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Figure 1: NE promotes lung tumor growth in vivo(a) Kaplan-Meier Survival curve for AdenoCre recipient LSL/K-ras/Elane+/+ and LSL/K-ras/Elane−/− mice; P=0.006, log-rank test. (b) Tumor area (%) for both groups at 8, 14, and 20 weeks post-AdenoCre. N=5 mice per group. Bars ± SEM. *P<0.01. (c) BALF neutrophil counts for AdenoCre recipient LSL/K-ras/Elane+/+ and LSL/K-ras/Elane−/− mice at the 14-week time point. N=5 mice per group. Bars ± SEM. P=NS. Representative H&E images at 14 weeks (d, e) and 20 weeks (f) post-AdenoCre. (g) Representative images for anti-p40phox (anti-neutrophil) IHC at 14 weeks post-AdenoCre. (h) Representative Ki-67 IHC 14 weeks post-AdenoCre. (i) Ki-67 (+) cells per tumor area for N=5 mice per group at the 14-week time point. Bars ± SEM. *P<0.01. (j) Representative IF images and quantification for pAkt and phospho-MEK/ERK from both groups of mice 14 weeks post-AdenoCre. Bars ± SEM. P<0.01 for pAkt.
Mentions: We subjected Lox—Stop—Lox K-rasG12D/Elane−/− (LSL-K-ras/Elane−/−) and control (LSL-K-ras/Elane+/+) mice to 5×106 pfu intratracheal adenoviral cre recombinase (AdenoCre) to activate mutant K-ras expression4. During the 28 weeks following AdenoCre administration, all LSL-K-ras/Elane+/+ but none of the LSL-K-ras/Elane−/− mice died. Survival analysis demonstrated a significant (P=0.006) advantage for LSL-K-ras/Elane−/− mice (Fig. 1a). NE-deficiency is not completely protective, as we have subsequently identified death beyond 30 weeks in independent studies. Tumor burden was markedly reduced in LSL-K-ras/Elane−/− mice at all time points (Fig. 1b,d–f). The differences observed represent a reduction in tumor growth and differentiation (less mature lesions), as tumor number was equivalent in the two groups (Supplementary Table 1). NE-mediated effects on tumor growth are not model specific, as similar reductions in tumor growth were observed in the Lewis Lung carcinoma model using WT and Elane−/− mice (Supplementary Fig. 1).

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