Introduction
Cellular senescence is a terminal cell proliferation arrest characterized by a distinct phenotype. Compared with their proliferating counterparts, senescent cells have enlarged volumes, display a flattened and vacuolated morphology, and express a variety of markers. The most widely used to identify senescent cells is senescence-associated β-galactosidase.
Cellular senescence can be triggered by a variety of stressors, including oncogenes, resulting in what is known as oncogene-induced senescence (OIS) [
1]. For example, expression of p95HER2, an oncogenic fragment of the tyrosine kinase receptor HER2, induces OIS in a variety of cell lines [
2].
The onset of senescence is characterized by a profound change in the secretome (i.e., all factors secreted by a given cell) that results in the so-called senescence-associated secretory phenotype or senescence secretome [
1]. Depending on the context, the senescence secretome has disparate effects. It may promote [
3] or impair [
4] immune surveillance against tumor cells in the liver and in the prostate, respectively. In fact, senescent cells may be short-lived or long-lived in vivo, in both immunocompetent [
3‐
5] and immunosuppressed [
2,
6] mice. Furthermore, the senescence secretome can suppress [
7] or promote [
8] tumor growth. These results can be rationalized assuming that the potent tumor suppressive effects of senescence can be reversed, particularly in advanced tumors, by modifying the composition of the senescence secretome and, thus, its effects on target cells.
Because the non-cell autonomous effects of senescent cells can suppress or contribute to tumor progression, the up- or downregulation of the senescence secretome could be a therapeutic strategy to treat cancer and perhaps many other diseases related to cellular senescence [
1]. Unfortunately, to date, there are no known strategies to regulate the production of the senescence secretome.
The proteolytic release of the extracellular domain of transmembrane proteins is known as ectodomain shedding. This type of limited proteolysis affects a diverse group of functionally unrelated transmembrane proteins, including membrane-anchored growth factors, cytokines, cell adhesion molecules, or transmembrane proteases [
9‐
12]. The proteases that cleave the vast majority of these transmembrane proteins are the metalloprotease disintegrins ADAM17 (also known as tumor necrosis factor-alpha-converting enzyme) or ADAM10 or both (reviewed in [
13]).
Some components frequently secreted by senescent cells, such as transmembrane epidermal growth factor (EGF)-like growth factors, are generated through ectodomain shedding. However, the contribution of ectodomain shedding to the senescence secretome remains largely unexplored. Although ADAM17 has been recently shown to be active in senescent cells [
14], its regulation or functional importance during senescence is unknown.
Here, we show that approximately 10 % of the components of the secretome of p95HER2-induced senescent cells are generated through the shedding of the ectodomains of membrane-anchored proteins. The main mechanism regulating the release of these ectodomains is the transcriptional regulation of the membrane-anchored precursors. Functional analysis shows that ADAM17 plays a major role in these cleavages. However, although ADAM17 protein levels increase during p95HER2-induced OIS, the activity of the metalloprotease does not increase, and this is likely because of the accumulation of cholesterol, a negative regulator of ADAM17, in senescent cells. Finally, we show that ADAM17 activity is required for several non-cell autonomous protumorigenic and prometastatic effects of p95HER2-induced senescent cells. Because the activity of ADAM17 can be pharmacologically downregulated, these results indicate that inhibition of this metalloprotease could be a means to target the undesired non-cell autonomous effects of cellular senescence.
Methods
Reagents
Doxycycline (Doxy.), phorbol myristate acetate, biotin, 1-10-phenanthroline, methyl-beta-cyclodextrin (MβCD), insulin, EGF, and hydrocortisone were from Sigma-Aldrich (St. Louis, MO, USA). Batimastat (BB94) was from Merck (Schwalbach, Germany).
Antibodies
Rabbit anti-phospho ERK (T202/Y204; #4370), anti-ERK (#9102), anti-phospho Akt (S473; #9271), anti-Akt (#9272), anti-phospho EGF receptor (EGFR) (Y1068; #3777), and anti-EGFR (#4267) were from Cell Signaling Technology (Danvers, MA, USA). Mouse anti-EpCAM (#sc-25308) was from Santa Cruz Biotechnologies (Santa Cruz, CA, USA), and mouse anti-pan actin (#MA5-11869) was from Thermo Scientific (Lafayette, CO, USA). Mouse anti-APP (#MAB348) and rabbit anti-ADAM17 (#AB19027) were from Millipore (Billerica, MA, USA). Rabbit anti-ADAM17 (#ab39162), rabbit anti-ADAM10 (#ab1997), goat anti-Met receptor (#ab10728), and mouse anti-glucose-6-phosphate (GPI) (#ab66340) antibodies were from Abcam (Cambridge, MA, USA). Rabbit anti-DDR1 (#10730) was from Sino Biological Inc. (Beijing, PR China), rabbit anti-GAPDH (#2275-PC-1) was from Trevigen (Gaithersburg, MD, USA), and mouse anti-HER2 (#MU-134-UC) was from BioGenex (San Ramon, CA, USA). Mouse anti-EphB4 (#AF446) was from R&D Systems (Minneapolis, MN, USA). Fluorochrome-conjugated antibodies were from Molecular Probes (Life Technologies, Grand Island, NY, USA).
Transcriptomic and proteomic analyses
Transcripts and secretome analysis were performed as described in [
15] and [
2], respectively. GeneChip expression probe array Gene Expression Omnibus reference number is GSE68256. Heatmap hierarchical clustering was performed as in [
16].
Plasmids
The expression vectors encoding alkaline phosphatase (AP)-tagged amphiregulin (AP-Areg), transforming growth factor-alpha (AP-TGF-α), and betacellulin (AP-BTC) were a kind gift from Shigeki Higashiyama (Department of Biochemistry and Molecular Genetics, Ehime University Graduate School of Medicine, Japan). Subcloning in the lentiviral vector pLex (Thermo Scientific) was performed by conventional molecular biology techniques. pGIPZ-based shRNA vectors targeting ADAM17 or non-silencing control were from Thermo Scientific. p95HER2 in pENTR1Dual (Invitrogen, Paisley, UK) was transferred into pINDUCER20 using LR Clonase II (Invitrogen). For control pINDUCER20 (pINDUCER20-Empty), cloramphenicol and ccdB genes were removed from pENTR1A Dual (5-SalI and 3’-XhoI) and the minimal MCS was transferred to pINDUCER20. pINDUCER20 was kindly provided by Stephen J Elledge (Howard Hughes Medical Institute, Chevy Chase, MD, USA) [
17].
Cell culture and infections
MCF7, MCF7 Tet-Off p95HER2, and MDA-MB-231-Luc were maintained as previously described [
2]. MCF10A was maintained in Dulbecco’s modified Eagle’s medium,/F-12, 10 % fetal bovine serum (FBS), 4 mmol/l L-glutamine (all from Gibco, Carlsbad, CA, USA), 9 μg/ml insulin, 0.5 μg/ml hydrocortisone, and 18 ng/ml EGF. MCF7 Tet-Off p95HER2 AP-Areg, AP-TGF-α, AP-BTC, shControl, and shADAM17 were generated by infecting MCF7 Tet-Off p95HER2 with the lentiviral particles obtained transfecting HEK293T cells with the plasmids described above (calcium phosphate method). Viral supernatant was applied to the cells in the presence of 8 μg/ml polybrene, and stable clones were selected with 1 μg/ml puromycin. MCF10A was infected with pINDUCER20-p95HER2 by using the same protocol, and selection was performed with 200 μg/mL G418. MCF10A infection with lentiviral vectors expressing AP-Areg and AP-TGF-α was performed with the plasmids and method indicated above.
Protein extraction and immunoblotting
Cells were lysed in 20 mM Tris-HCl pH 7.4, 137 mM NaCl, 2 mM EDTA pH 8.0, 10 % glycerol, 1 % NP-40, protease inhibitor cocktail (Roche, Penzberg, Germany), 1.3 mM sodium orthovanadate, 10 mM 1-10-phenanthroline, and 1 μM BB94. Cell lysates were quantified by using the Pierce BCA Protein Assay kit (Pierce, Rockford, IL, USA), and equal amounts of protein were separated on SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. These were blocked with 5 % bovine serum albumin or skim milk in TBS-Tween 0.1 % and then incubated with primary antibodies, and bound antibodies were detected with the appropriate peroxidase-conjugated secondary antibodies (Amersham GE Healthcare, Piscataway, NJ, USA) by using the ECL detection system (Millipore). To detect ADAM17, the same amount of protein from cell lysates was concentrated with wheat germ agglutinin (WGA)-agarose beads (Vector Laboratories, Peterborough, UK) for 2 h at 4 °C. Proteins were eluted directly in SDS-polyacrylamide gel electrophoresis loading buffer.
Determination of proteins in the collected conditioned media was performed following the same protocol after concentrating 100× the samples by using centricons (Amicon Ultracel 3K; Millipore). Densitometric quantification was performed by using ImageJ software.
Enzyme-linked immunosorbent assay
Cells were plated and the next day were washed twice with 1X phosphate-buffered saline (PBS), and medium was changed to serum-free medium with L-glutamine. Conditioned media were collected 48 h later, spun down at 200×g for 5 min, and transferred to clean tubes. Amphiregulin was determined in accordance with the instructions of the manufacturer (RayBiotech, Norcross, GA, USA).
mRNA expression
RNA was isolated by using an RNeasy Mini kit (Qiagen, Hilden, Germany) and reverse-transcribed by using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Weiterstadt, Germany) in accordance with the instructions of the manufacturers. Quantitative reverse transcription-polymerase chain reaction was performed by using Taqman primers (Applied Biosystems) for ADAM17 (Hs01041915_m1), ADAM10 (Hs00153853_m1), and GAPDH (Hs03929097_g1).
Biotin labeling
Cells were washed three times with ice-cold 1X PBS pH 8.0 and labeled with 1 mg/ml sulfo-NHS-LC-biotin (Pierce) in 1X PBS pH 8.0 gently shaking for 1 h at 4 °C. Biotin was inactivated by washing with 50 mM Tris pH 7.4, and cells were extensively washed with 1X PBS. Then cells were harvested and lysed, and immunoprecipitation was performed by using an anti-ADAM17 antibody or a control IgG. Immunoprecipitates were analyzed by Western blotting by using streptavidin-POD conjugate (Roche) or anti-ADAM17 antibody (Abcam).
Confocal microscopy
Cells were seeded on glass coverslips, washed with 1X PBS, fixed with 4 % formaldehyde for 20 min, and permeabilized with 0.2 % Triton X-100 for 10 min. 1X PBS containing 1 % bovine serum albumin, 0.1 % saponin, and 0.02 % NaN3 was used for blocking (1 h), primary antibody binding (2 h), and secondary fluorochrome-conjugated antibody binding (40 min in dark). Preparations were mounted by using Vectashield with DAPI (4’,6’-diamidino-2-phenylindole) (Vector Laboratories). All procedures were performed at room temperature. Images were captured by using an Olympus FV1000 confocal microscope (Olympus Corporation, Tokyo, Japan).
Ectodomain shedding assay
MCF7 Tet-Off p95HER2 cells were induced for 5 days and replated at the same cell concentration. Next day, cells were washed with Opti-MEM (Invitrogen) without serum or growth factors, and the medium was replaced 1 h later with fresh Opti-MEM with or without the indicated stimuli. Cells and supernatants were harvested at the designated time points, and AP activity was measured at an absorbance of 405 nm after incubation with the AP substrate 4-nitrophenyl phosphate (Sigma-Aldrich). At least three wells per condition were performed, and the ratio between the AP activity in the supernatant and the cell lysate plus supernatant was calculated. The fold increase in the ratio of AP activity relative to the control is shown.
Proliferation assays
Proliferation was analyzed by cell counting. After trypsinization, viable cells determined by trypan blue dye exclusion were counted on a Neubauer chamber.
Senescence-associated β-galactosidase activity
Cells were plated on coverslips and analyzed by using a senescence β-galactosidase staining kit (Cell Signaling Technology) in accordance with the indications of the manufacturer.
Filipin staining
Cells were washed three times with 1X PBS and fixed with 3 % paraformaldehyde for 1 h at room temperature. Washes were repeated, and cells were incubated with 1.5 mg/ml glycine in 1X PBS for 10 minutes. Staining was performed for 2 h with 0.05 mg/ml filipin complex (Sigma-Aldrich) in 1X PBS/10 % FBS. After the cells were rinsed three times with 1X PBS, they were analyzed by fluorescence microscopy (BX61 Olympus).
Migration assays
Cell migration was determined with 24-well format Boyden chambers containing FluoroBlok PET membranes with 8-μm pores (BD Biosciences, Heidelberg, Germany). Conditioned media were obtained after incubating cells with serum-free medium with glutamine for 48 h, spun down, and concentrated 100 times by using centricons (Amicon Ultracel 3K). Concentrated conditioned media were diluted 10 times in serum-free medium and added to the bottom part of the Boyden chambers. MDA-MB-231 Luc or MCF7 cells were plated in the upper chambers and migration ability was evaluated 24 or 48 h later, respectively, by staining the cells with 1 μM SYTO-9 (Life Technologies) and counting them on the underside of the filters by using fluorescence microscopy.
Mouse model of breast cancer metastasis
MCF7 Tet-Off p95HER2 cells expressing a short hairpin control or targeting ADAM17 were co-injected with MDA-MB-231 Luc cells into the right flanks of 6- to 8-week-old female BALB/c athymic mice (Charles River Laboratories, Paris, France) in addition to a 17β-estradiol pellet (Innovative Research of America, Sarasota, FL, USA). The expression of p95HER2 was repressed by adding doxycycline (50 mg/kg per day) to the drinking water until tumors were about 100 mm3. Tumor xenografts were measured with calipers every 3 days, and tumor volume was determined by using the formula: (length × width2) × (pi/6). Tumors were resected when they reached 300 mm3, and metastatic colonization was monitored by in vivo bioluminescence imaging with the IVIS-200 imaging system (PerkinElmer, Waltham, MA, USA). At the end of the experiment, animals were anesthetized with a 1.5 % isofluorane-air mixture and were sacrificed by cervical dislocation. Mice were maintained and treated in accordance with institutional guidelines of Vall d’Hebron University Hospital Care and Use Committee.
Statistics
Data are presented as average ± standard deviation and were analyzed by the two-sided Student t test. Results were considered to be statistically significant at P value of less than 0.05. All statistical analyses were conducted by using the GraphPad Prism 5 Statistical Software (GraphPad Software, Inc., La Jolla, CA, USA).
Discussion
In this work, we show that approximately one tenth of the components of the senescence secretome are generated through protein ectodomain shedding (Additional file
1: Figure. S1c) and that one third of these are cleaved by ADAM17. This report is the first functional analysis of the contribution of ADAM17-mediated ectodomain shedding to the non-cell autonomous effects of oncogene-induced senescent cells.
The regulation of ADAM17 during senescence is complex. One of the intracellular pathways that activates ADAM17 is the MEK-ERK pathway (reviewed in [
35]). Thus, one could assume that ADAM17 is activated in p95HER2-induced senescent cells, where the ERK1,2 pathway is constitutively active [
15] (Figs.
2a and
4b). In fact, a recent report shows that ADAM17 is activated in Ras-induced senescent cells [
14]. However, our results clearly show that activation of p95HER2 does not result in ADAM17 activation. In fact, p95HER2-induced senescent cells accumulate partially inactive ADAM17. This restriction of ADAM17 activity is likely due to the accumulation of cholesterol in senescent cells. This result contrasts with that published by Effenberger et al., who showed similar levels of ADAM17 in PC3 proliferating cells and in the same cells after induction of senescence with doxorubicin [
14]. The likely explanation(s) for these apparently disparate observation may reside in differences in the cell type (PC3 and MCF7 are derived from prostate and breast cancers, respectively) or in the trigger of senescence (DNA-damage induced by Doxorubicin versus expression of p95HER2) or in both. For instance, ADAM17 levels and activity were differentially regulated in MCF7 and MCF10, which are both of breast origin and express the same oncogene.
Although the exact mechanism of ADAM17 inhibition is not known, it seems to be related to the compartmentalization of the metalloproteinase in plasma membrane subdomains where shedding substrates are not accessible [
25‐
30]. In addition to increasing the activity of ADAM17, MβCD decreased the levels of the metalloprotease, particularly those of the processed form (Fig.
4b). A way to interpret this result is by assuming that ADAM17 inhibited by cholesterol has a longer half-life than active ADAM17. Future work will be directed to clarify whether these interpretations of the results explain not only the upregulation of the levels of ADAM17 and the restriction of its activity in p95HER2-induced senescent cells but the differences observed between p95HER2- and Ras-induced senescent cells as well.
The clear correlation between the levels of the transcripts encoding shedding substrates and the levels of ectodomains in the senescence secretome (Fig.
1d) indicates that the activity of ADAM17 is limiting in senescent cells: only substrates whose expression increases, such as Met or Areg, are cleaved. Under these limiting conditions, substrates whose expression does not increase during senescence, such as APP, are probably outcompeted and, as a result, their shedding decreases during OIS.
Despite this restriction, the remaining activity of ADAM17 clearly contributes to the protumorigenic effects of p95HER2-induced senescent cells. The data in Fig.
5a show that factors whose secretion depends on ADAM17 increase cell motility. This result, along with the fact that the effect of ADAM17 is non-cell autonomous (Fig.
5c-
f), led us to conclude that the proteolytic activity of ADAM17 acts on factors that, when released, increase the metastatic ability of cancer cells.
Given the lack of modulators of the senescence secretome and the fact that the activity of ADAM17 can be upregulated by different compounds or inhibited with small-molecule synthetic inhibitors (reviewed in [
36]) or monoclonal antibodies [
37], our results open up a possibility that part of the effects of the senescence secretome can be modulated by regulating the activity of ADAM17. Thus, the pharmacological modulation of ADAM17 may represent a means to target the non-cell autonomous effects of cellular senescence, which may contribute to different diseases, including cancer [
1].
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
BM helped to design the study, analyze the data, and prepare the manuscript; confirmed the secretome analysis; and performed the in vitro and in vivo experiments involving ADAM17 and MβCD-related assays. AM-B helped to design the study, analyze the data, and prepare the manuscript; provided input on secretome analysis; performed the secretome of MCF7 Tet-Off p95HER2 cells and the in vitro experiments involving ADAM10 and MβCD-related assays; generated and characterized MCF10A Tet-On p95HER2 cells. JV designed and analyzed the label-free proteomic studies and critically revised the manuscript. JA designed the study, analyzed the data, and prepared the manuscript. All authors read and approved the final manuscript..