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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 co-localizes with and degrades IRS-1(a) IRS-1 Western blot following incubation of recombinant IRS-1 protein with NE over a range of molar ratios. (b) IRS-1 and β-actin Western blots for NE-exposed A549 cell lysates. (c) 3H uptake for IRS-1 silenced (or SCR control) A549 cells subsequently exposed to NE or vehicle. Results from a representative experiment in triplicate. Bars ± SEM. *P<0.001 from NE=0 control. **P<0.05 from NE=80. Inset, IRS-1 blot of SCR and IRS-1 siRNA treated lysates. (d) 3H uptake for IRS-1 over-expressing A549 cells subsequently exposed to NE or vehicle. Results from a representative experiment in triplicate. Bars ± SEM. *P<0.01. Inset, IRS-1 blot of IRS-1 vector and control treated lysates. (e) Confocal images for IRS-1 and NE from A549 cells exposed to NE (or vehicle). (f) Representative IRS-1 IHC images from LSL/K-ras/Elane+/+ and LSL/K-ras/Elane−/− mice 14 weeks post-AdenoCre. (g) IRS-1 Western blot for 14-week post-AdenoCre tumor homogenates from both groups (N=5). Results presented as relative band density ± SEM. P<0.001. (h) IRS-1 real-time PCR for 14-week post-AdenoCre tumor homogenates from both groups (N=4). Results expressed as GAPDH CT/IRS-1 CT ± SEM. (i) Representative IF images for pSer and pTyr IRS-1 from LSL/K-ras/Elane+/+ and LSL/K-ras/Elane−/− tumors 14-wks post-AdenoCre. (j) Representative images of human lung adenocarcinoma specimens for NE=2 and IRS-1=0 (Case 121t) and NE=0 and IRS-1=3 (Case 649t). NE and IRS-1 were considered discordant when either one was present but the other absent/faint in the same view. The empirical probability of discordance was 0.88, which was significantly greater than chance (0.5), P<0.001.
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Figure 4: NE co-localizes with and degrades IRS-1(a) IRS-1 Western blot following incubation of recombinant IRS-1 protein with NE over a range of molar ratios. (b) IRS-1 and β-actin Western blots for NE-exposed A549 cell lysates. (c) 3H uptake for IRS-1 silenced (or SCR control) A549 cells subsequently exposed to NE or vehicle. Results from a representative experiment in triplicate. Bars ± SEM. *P<0.001 from NE=0 control. **P<0.05 from NE=80. Inset, IRS-1 blot of SCR and IRS-1 siRNA treated lysates. (d) 3H uptake for IRS-1 over-expressing A549 cells subsequently exposed to NE or vehicle. Results from a representative experiment in triplicate. Bars ± SEM. *P<0.01. Inset, IRS-1 blot of IRS-1 vector and control treated lysates. (e) Confocal images for IRS-1 and NE from A549 cells exposed to NE (or vehicle). (f) Representative IRS-1 IHC images from LSL/K-ras/Elane+/+ and LSL/K-ras/Elane−/− mice 14 weeks post-AdenoCre. (g) IRS-1 Western blot for 14-week post-AdenoCre tumor homogenates from both groups (N=5). Results presented as relative band density ± SEM. P<0.001. (h) IRS-1 real-time PCR for 14-week post-AdenoCre tumor homogenates from both groups (N=4). Results expressed as GAPDH CT/IRS-1 CT ± SEM. (i) Representative IF images for pSer and pTyr IRS-1 from LSL/K-ras/Elane+/+ and LSL/K-ras/Elane−/− tumors 14-wks post-AdenoCre. (j) Representative images of human lung adenocarcinoma specimens for NE=2 and IRS-1=0 (Case 121t) and NE=0 and IRS-1=3 (Case 649t). NE and IRS-1 were considered discordant when either one was present but the other absent/faint in the same view. The empirical probability of discordance was 0.88, which was significantly greater than chance (0.5), P<0.001.

Mentions: NE rapidly hydrolyzed IRS1 at 1:100 molar concentrations (Fig. 4a). Cell proliferative concentrations of NE eliminated IRS1 within A549 cells (Fig. 4b). Silencing of IRS1 gene expression induced tumor cell proliferation (Fig. 4c). Marked IRS1 over-expression reduced tumor cell growth and abrogated the proliferative effects of NE, confirming that IRS1 loss is a required event in this process (Fig. 4d). Hence, independent of NE, IRS1 is capable of regulating tumor cell proliferation.


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 co-localizes with and degrades IRS-1(a) IRS-1 Western blot following incubation of recombinant IRS-1 protein with NE over a range of molar ratios. (b) IRS-1 and β-actin Western blots for NE-exposed A549 cell lysates. (c) 3H uptake for IRS-1 silenced (or SCR control) A549 cells subsequently exposed to NE or vehicle. Results from a representative experiment in triplicate. Bars ± SEM. *P<0.001 from NE=0 control. **P<0.05 from NE=80. Inset, IRS-1 blot of SCR and IRS-1 siRNA treated lysates. (d) 3H uptake for IRS-1 over-expressing A549 cells subsequently exposed to NE or vehicle. Results from a representative experiment in triplicate. Bars ± SEM. *P<0.01. Inset, IRS-1 blot of IRS-1 vector and control treated lysates. (e) Confocal images for IRS-1 and NE from A549 cells exposed to NE (or vehicle). (f) Representative IRS-1 IHC images from LSL/K-ras/Elane+/+ and LSL/K-ras/Elane−/− mice 14 weeks post-AdenoCre. (g) IRS-1 Western blot for 14-week post-AdenoCre tumor homogenates from both groups (N=5). Results presented as relative band density ± SEM. P<0.001. (h) IRS-1 real-time PCR for 14-week post-AdenoCre tumor homogenates from both groups (N=4). Results expressed as GAPDH CT/IRS-1 CT ± SEM. (i) Representative IF images for pSer and pTyr IRS-1 from LSL/K-ras/Elane+/+ and LSL/K-ras/Elane−/− tumors 14-wks post-AdenoCre. (j) Representative images of human lung adenocarcinoma specimens for NE=2 and IRS-1=0 (Case 121t) and NE=0 and IRS-1=3 (Case 649t). NE and IRS-1 were considered discordant when either one was present but the other absent/faint in the same view. The empirical probability of discordance was 0.88, which was significantly greater than chance (0.5), P<0.001.
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Figure 4: NE co-localizes with and degrades IRS-1(a) IRS-1 Western blot following incubation of recombinant IRS-1 protein with NE over a range of molar ratios. (b) IRS-1 and β-actin Western blots for NE-exposed A549 cell lysates. (c) 3H uptake for IRS-1 silenced (or SCR control) A549 cells subsequently exposed to NE or vehicle. Results from a representative experiment in triplicate. Bars ± SEM. *P<0.001 from NE=0 control. **P<0.05 from NE=80. Inset, IRS-1 blot of SCR and IRS-1 siRNA treated lysates. (d) 3H uptake for IRS-1 over-expressing A549 cells subsequently exposed to NE or vehicle. Results from a representative experiment in triplicate. Bars ± SEM. *P<0.01. Inset, IRS-1 blot of IRS-1 vector and control treated lysates. (e) Confocal images for IRS-1 and NE from A549 cells exposed to NE (or vehicle). (f) Representative IRS-1 IHC images from LSL/K-ras/Elane+/+ and LSL/K-ras/Elane−/− mice 14 weeks post-AdenoCre. (g) IRS-1 Western blot for 14-week post-AdenoCre tumor homogenates from both groups (N=5). Results presented as relative band density ± SEM. P<0.001. (h) IRS-1 real-time PCR for 14-week post-AdenoCre tumor homogenates from both groups (N=4). Results expressed as GAPDH CT/IRS-1 CT ± SEM. (i) Representative IF images for pSer and pTyr IRS-1 from LSL/K-ras/Elane+/+ and LSL/K-ras/Elane−/− tumors 14-wks post-AdenoCre. (j) Representative images of human lung adenocarcinoma specimens for NE=2 and IRS-1=0 (Case 121t) and NE=0 and IRS-1=3 (Case 649t). NE and IRS-1 were considered discordant when either one was present but the other absent/faint in the same view. The empirical probability of discordance was 0.88, which was significantly greater than chance (0.5), P<0.001.
Mentions: NE rapidly hydrolyzed IRS1 at 1:100 molar concentrations (Fig. 4a). Cell proliferative concentrations of NE eliminated IRS1 within A549 cells (Fig. 4b). Silencing of IRS1 gene expression induced tumor cell proliferation (Fig. 4c). Marked IRS1 over-expression reduced tumor cell growth and abrogated the proliferative effects of NE, confirming that IRS1 loss is a required event in this process (Fig. 4d). Hence, independent of NE, IRS1 is capable of regulating tumor cell proliferation.

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