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Role of ADAM17 in the non-cell autonomous effects of oncogene-induced senescence.

Morancho B, Martínez-Barriocanal Á, Villanueva J, Arribas J - Breast Cancer Res. (2015)

Bottom Line: Considering these findings, we conclude that ectodomain shedding is tightly regulated in oncogene-induced senescent cells by integrating transcription of the shedding substrates with limiting ADAM17 activity.The remaining activity of ADAM17 contributes to the non-cell autonomous protumorigenic effects of p95HER2-induced senescent cells.Because ADAM17 is druggable, these results represent an approximation to the pharmacological regulation of the senescence secretome.

View Article: PubMed Central - PubMed

Affiliation: Preclinical Research Program, Vall d'Hebron Institute of Oncology (VHIO), Psg. Vall d'Hebron 119-129, Barcelona, 08035, Spain. bmorancho@vhio.net.

ABSTRACT

Introduction: Cellular senescence is a terminal cell proliferation arrest that can be triggered by oncogenes. One of the traits of oncogene-induced senescence (OIS) is the so-called senescence-associated secretory phenotype or senescence secretome. Depending on the context, the non-cell autonomous effects of OIS may vary from tumor suppression to promotion of metastasis. Despite being such a physiological and pathologically relevant effector, the mechanisms of generation of the senescence secretome are largely unknown.

Methods: We analyzed by label-free proteomics the secretome of p95HER2-induced senescent cells and compared the levels of the membrane-anchored proteins with their transcript levels. Then, protein and RNA levels of ADAM17 were evaluated by using Western blot and reverse transcription-polymerase chain reaction, its localization by using biotin labeling and immunofluorescence, and its activity by using alkaline phosphatase-tagged substrates. The p95HER2-expressing cell lines, senescent MCF7 and proliferating MCF10A, were analyzed to study ADAM17 regulation. Finally, we knocked down ADAM17 to determine its contribution to the senescence-associated secretome. The effect of this secretome was evaluated in migration assays in vitro and in nude mice by assessing the metastatic ability of orthotopically co-injected non-senescent cells.

Results: Using breast cancer cells expressing p95HER2, a constitutively active fragment of the proto-oncogene HER2 that induces OIS, we show that the extracellular domains of a variety of membrane-bound proteins form part of the senescence secretome. We determine that these proteins are regulated transcriptionally and, in addition, that their shedding is limited by the protease ADAM17. The activity of the sheddase is constrained, at least in part, by the accumulation of cellular cholesterol. The blockade of ADAM17 abrogates several prometastatic effects of the p95HER2-induced senescence secretome, both in vitro and in vivo.

Conclusions: Considering these findings, we conclude that ectodomain shedding is tightly regulated in oncogene-induced senescent cells by integrating transcription of the shedding substrates with limiting ADAM17 activity. The remaining activity of ADAM17 contributes to the non-cell autonomous protumorigenic effects of p95HER2-induced senescent cells. Because ADAM17 is druggable, these results represent an approximation to the pharmacological regulation of the senescence secretome.

No MeSH data available.


Related in: MedlinePlus

Effect of cholesterol depletion on the protein levels and activity of ADAM17 in p95HER2-induced senescent cells. a MCF7 Tet-Off p95HER2 cells were cultured with or without doxycycline for 5 days and further incubated in serum-free media with or without 1 mM MβCD for 2 additional days. Then cells were stained with filipin. Representative bright-field and filipin staining images are shown. b The same cells as in (a) were treated with different concentrations of MβCD. Control and treated cells were harvested and lysed, and cell lysates were analyzed by Western blot with the indicated antibodies. The densitometric quantification of three independent experiments is expressed as average ± standard deviation. *P <0.05 using the two-sided Student’s t test. c MCF7 Tet-Off p95HER2 or MCF10A Tet-On p95HER2 cells expressing AP-tagged TGF-α (left panels), Areg (middle panels), or BTC (right panel) were treated with or without doxycycline and MβCD or vehicle for 48 h as indicated. AP was quantified in serum-free conditioned media and cell lysates. Data shown represent the averages and standard deviations of three independent experiments. *P < 0.05 using the two-sided Student’s t test. AP alkaline phosphatase, BTC betacellulin, Doxy doxycycline, MβCD methyl-beta-cyclodextrin, TGF transforming growth factor
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Fig4: Effect of cholesterol depletion on the protein levels and activity of ADAM17 in p95HER2-induced senescent cells. a MCF7 Tet-Off p95HER2 cells were cultured with or without doxycycline for 5 days and further incubated in serum-free media with or without 1 mM MβCD for 2 additional days. Then cells were stained with filipin. Representative bright-field and filipin staining images are shown. b The same cells as in (a) were treated with different concentrations of MβCD. Control and treated cells were harvested and lysed, and cell lysates were analyzed by Western blot with the indicated antibodies. The densitometric quantification of three independent experiments is expressed as average ± standard deviation. *P <0.05 using the two-sided Student’s t test. c MCF7 Tet-Off p95HER2 or MCF10A Tet-On p95HER2 cells expressing AP-tagged TGF-α (left panels), Areg (middle panels), or BTC (right panel) were treated with or without doxycycline and MβCD or vehicle for 48 h as indicated. AP was quantified in serum-free conditioned media and cell lysates. Data shown represent the averages and standard deviations of three independent experiments. *P < 0.05 using the two-sided Student’s t test. AP alkaline phosphatase, BTC betacellulin, Doxy doxycycline, MβCD methyl-beta-cyclodextrin, TGF transforming growth factor

Mentions: Previous reports have shown that ADAM17 is inhibited by high cholesterol levels [25–30]. Senescent cells tend to accumulate cholesterol [31]; in fact, it has been previously shown that HER2 (NeuT)-induced senescence results in a marked accumulation of cellular cholesterol [32]. Thus, we reasoned that the high levels of cholesterol in p95HER2-induced senescent cells may lead to the accumulation of partially inactive ADAM17. As expected, treatment with MβCD, a compound that selectively extracts membrane cholesterol, reduced the levels of cholesterol in p95HER2-senescent cells (Fig. 4a).Fig. 4


Role of ADAM17 in the non-cell autonomous effects of oncogene-induced senescence.

Morancho B, Martínez-Barriocanal Á, Villanueva J, Arribas J - Breast Cancer Res. (2015)

Effect of cholesterol depletion on the protein levels and activity of ADAM17 in p95HER2-induced senescent cells. a MCF7 Tet-Off p95HER2 cells were cultured with or without doxycycline for 5 days and further incubated in serum-free media with or without 1 mM MβCD for 2 additional days. Then cells were stained with filipin. Representative bright-field and filipin staining images are shown. b The same cells as in (a) were treated with different concentrations of MβCD. Control and treated cells were harvested and lysed, and cell lysates were analyzed by Western blot with the indicated antibodies. The densitometric quantification of three independent experiments is expressed as average ± standard deviation. *P <0.05 using the two-sided Student’s t test. c MCF7 Tet-Off p95HER2 or MCF10A Tet-On p95HER2 cells expressing AP-tagged TGF-α (left panels), Areg (middle panels), or BTC (right panel) were treated with or without doxycycline and MβCD or vehicle for 48 h as indicated. AP was quantified in serum-free conditioned media and cell lysates. Data shown represent the averages and standard deviations of three independent experiments. *P < 0.05 using the two-sided Student’s t test. AP alkaline phosphatase, BTC betacellulin, Doxy doxycycline, MβCD methyl-beta-cyclodextrin, TGF transforming growth factor
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4532141&req=5

Fig4: Effect of cholesterol depletion on the protein levels and activity of ADAM17 in p95HER2-induced senescent cells. a MCF7 Tet-Off p95HER2 cells were cultured with or without doxycycline for 5 days and further incubated in serum-free media with or without 1 mM MβCD for 2 additional days. Then cells were stained with filipin. Representative bright-field and filipin staining images are shown. b The same cells as in (a) were treated with different concentrations of MβCD. Control and treated cells were harvested and lysed, and cell lysates were analyzed by Western blot with the indicated antibodies. The densitometric quantification of three independent experiments is expressed as average ± standard deviation. *P <0.05 using the two-sided Student’s t test. c MCF7 Tet-Off p95HER2 or MCF10A Tet-On p95HER2 cells expressing AP-tagged TGF-α (left panels), Areg (middle panels), or BTC (right panel) were treated with or without doxycycline and MβCD or vehicle for 48 h as indicated. AP was quantified in serum-free conditioned media and cell lysates. Data shown represent the averages and standard deviations of three independent experiments. *P < 0.05 using the two-sided Student’s t test. AP alkaline phosphatase, BTC betacellulin, Doxy doxycycline, MβCD methyl-beta-cyclodextrin, TGF transforming growth factor
Mentions: Previous reports have shown that ADAM17 is inhibited by high cholesterol levels [25–30]. Senescent cells tend to accumulate cholesterol [31]; in fact, it has been previously shown that HER2 (NeuT)-induced senescence results in a marked accumulation of cellular cholesterol [32]. Thus, we reasoned that the high levels of cholesterol in p95HER2-induced senescent cells may lead to the accumulation of partially inactive ADAM17. As expected, treatment with MβCD, a compound that selectively extracts membrane cholesterol, reduced the levels of cholesterol in p95HER2-senescent cells (Fig. 4a).Fig. 4

Bottom Line: Considering these findings, we conclude that ectodomain shedding is tightly regulated in oncogene-induced senescent cells by integrating transcription of the shedding substrates with limiting ADAM17 activity.The remaining activity of ADAM17 contributes to the non-cell autonomous protumorigenic effects of p95HER2-induced senescent cells.Because ADAM17 is druggable, these results represent an approximation to the pharmacological regulation of the senescence secretome.

View Article: PubMed Central - PubMed

Affiliation: Preclinical Research Program, Vall d'Hebron Institute of Oncology (VHIO), Psg. Vall d'Hebron 119-129, Barcelona, 08035, Spain. bmorancho@vhio.net.

ABSTRACT

Introduction: Cellular senescence is a terminal cell proliferation arrest that can be triggered by oncogenes. One of the traits of oncogene-induced senescence (OIS) is the so-called senescence-associated secretory phenotype or senescence secretome. Depending on the context, the non-cell autonomous effects of OIS may vary from tumor suppression to promotion of metastasis. Despite being such a physiological and pathologically relevant effector, the mechanisms of generation of the senescence secretome are largely unknown.

Methods: We analyzed by label-free proteomics the secretome of p95HER2-induced senescent cells and compared the levels of the membrane-anchored proteins with their transcript levels. Then, protein and RNA levels of ADAM17 were evaluated by using Western blot and reverse transcription-polymerase chain reaction, its localization by using biotin labeling and immunofluorescence, and its activity by using alkaline phosphatase-tagged substrates. The p95HER2-expressing cell lines, senescent MCF7 and proliferating MCF10A, were analyzed to study ADAM17 regulation. Finally, we knocked down ADAM17 to determine its contribution to the senescence-associated secretome. The effect of this secretome was evaluated in migration assays in vitro and in nude mice by assessing the metastatic ability of orthotopically co-injected non-senescent cells.

Results: Using breast cancer cells expressing p95HER2, a constitutively active fragment of the proto-oncogene HER2 that induces OIS, we show that the extracellular domains of a variety of membrane-bound proteins form part of the senescence secretome. We determine that these proteins are regulated transcriptionally and, in addition, that their shedding is limited by the protease ADAM17. The activity of the sheddase is constrained, at least in part, by the accumulation of cellular cholesterol. The blockade of ADAM17 abrogates several prometastatic effects of the p95HER2-induced senescence secretome, both in vitro and in vivo.

Conclusions: Considering these findings, we conclude that ectodomain shedding is tightly regulated in oncogene-induced senescent cells by integrating transcription of the shedding substrates with limiting ADAM17 activity. The remaining activity of ADAM17 contributes to the non-cell autonomous protumorigenic effects of p95HER2-induced senescent cells. Because ADAM17 is druggable, these results represent an approximation to the pharmacological regulation of the senescence secretome.

No MeSH data available.


Related in: MedlinePlus