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The fibronectin III-1 domain activates a PI3-Kinase/Akt signaling pathway leading to αvβ5 integrin activation and TRAIL resistance in human lung cancer cells
© The Author(s). 2016
- Received: 15 January 2016
- Accepted: 26 July 2016
- Published: 2 August 2016
Fibronectin is a mechanically sensitive protein which is organized in the extracellular matrix as a network of interacting fibrils. The lung tumor stroma is enriched for fibronectin which is thought to contribute to metastasis and drug resistance. Fibronectin is an elastic, multi-modular protein made up of individually folded domains, some of which can stretch in response to increased mechanical tension. Very little is known about the relationship of fibronectin’s unfolded domains to lung cancer resistance to chemotherapy. In the present study, we evaluated the impact of unfolding the first Type III domain of fibronectin (FnIII-1c) on TNF-related apoptosis inducing ligand (TRAIL) resistance.
NCI-H460 non-small cell lung cancer cells were treated with FnIII-1c then assessed for TRAIL-induced apoptosis. Subsequent analysis of FnIII-1c-mediated signaling pathways was also completed. Human non-small cell lung cancer tissue sections were assessed for the expression of vitronectin by immunohistochemistry.
FnIII-1c inhibited TRAIL-induced activation of caspase 8 and subsequent apoptosis in NCI-H460 lung cancer cells. FnIII-1c treatment was associated with the activation of the phosphatidylinositol-3-kinase/alpha serine/threonine kinase (PI3K/Akt) pathway and the αvβ5 integrin receptor for vitronectin, both of which were required for TRAIL resistance. Immunohistochemical staining of sections from non-small cell lung cancers showed that vitronectin was localized around blood vessels and in the tumor-stroma interface.
Unfolding of Type III domains within the fibronectin matrix may promote TRAIL resistance through the activation of a PI3K/Akt/αvβ5 signaling axis and point to a novel mechanism by which changes in secondary structure of fibronectin contribute to cancer cell resistance to apoptosis.
Cancers develop in a mechanically and biologically active microenvironment that continuously evolves with the disease. The tumor microenvironment is desmoplastic – abundant in infiltrating immune cells, tumor-associated fibroblasts and fibrotic extracellular matrix (ECM) proteins – and this “reactive” stroma distinguishes carcinomas from normal tissues. In addition to desmoplasia, the tumor stroma is characterized by deregulated ECM remodeling and tissue stiffening, which are associated with malignant progression .
TNF-related apoptosis inducing ligand (TRAIL) is a novel therapeutic agent currently under clinical trial for the treatment of non-small cell lung cancer (NSCLC) . TRAIL binds to death receptors 4 and 5 (DR4, DR5) to induce apoptosis through the extrinsic pathway. Binding of trimeric TRAIL to DR4/5 stimulates receptor oligomerization and the formation of the death inducing signaling complex (DISC). The components of the DISC include Fas-associated protein with death domain (FADD), caspase 8, and cellular FLICE-like inhibitor protein (c-FLIP). Proper formation of the DISC results in the activation and cleavage of caspase 8, which then initiates the apoptotic death program . Preclinical studies implicated TRAIL as an ideal therapy for non-small cell lung cancer (NSCLC). In mouse models of human lung cancer, TRAIL promoted tumor regression, delayed tumor growth, and improved overall survival . In addition, late stage human tumors stained positively for DR4 (99 %) and DR5 (82 %) , suggesting that those tumors could be targeted with TRAIL based therapeutics. However, results from clinical trials using DR4 or DR5 agonists in combination with traditional chemotherapies showed no improvement in response rates or progression free survival (PGS) . The failure to translate preclinical success in clinical trials suggests a need for a deeper investigation of the mechanisms regulating death receptor function.
Fibronectin is one of the most common and abundant ECM proteins deposited in the stroma of aggressive tumors [6–8]. In the metastatic niche, fibronectin functions as a scaffold for the continued recruitment of haematopoietic and invading cancer cells . In NSCLC, fibronectin overexpression is associated with increased angiogenesis, enhanced cancer cell survival, and metastasis . Fibronectin is a mechanically sensitive protein whose secondary structure is organized into individually folded domains termed the type I, II and III . Unlike the type I and II domains, fibronectin type III domains lack stabilizing disulfide bonds which allows them to unfold in response to mechanical and cell-contractile forces which are generated in response to increased tissue rigidity [12–15]. Recent studies have shown that tumor-associated fibronectin matrices are stiffer and the fibronectin fibers stretched and unfolded . Very little is known about the impact of these changes in fibronectin secondary structure on either tumor progression or chemoresistance.
Atomic force microscopy and steered molecular dynamics have identified a partially unfolded, stable intermediate of the first type III domain of fibronectin (FnIII-1c) which is predicted to form in response to contractile unfolding . In this study, we investigated the impact of the unfolded FnIII-1 on TRAIL-induced apoptosis in NSCLC cells using the FnIII-1c peptide to recapitulate the unfolded FnIII-1 structure . We found that FnIII-1c inhibited TRAIL-induced apoptosis via a PI3K-Akt dependent activation of integrin αvβ5. Additionally, we detected vitronectin, the ligand for integrin αvβ5, in human NSCLC tumors surrounding blood vessels and in the interstitium between the tumor and stroma. Our data suggest that the changes in fibronectin secondary structure, which occur in response to the increased tissue rigidity of the tumor stroma, may contribute to apoptosis resistance.
Antibodies and reagents
All reagents were purchased from Sigma (St. Louis, MO) unless indicated otherwise. Fetal bovine serum (FBS) was from Hyclone (Logan, UT). Recombinant fibronectin type III domains FnIII-1c, FnIII-10n and FnIII-13 were generated and purified as previously described . Recombinant human TRAIL/TNFSF10 was purchased from R&D systems (Minneapolis, MN). The PI3K inhibitor LY294002 was purchased from Biomol (Plymouth Meeting, PA) and the Akt inhibitor VIII (AG730) was purchased from Sigma. Polyclonal rabbit antibody against phospho-Akt (Ser473), Hoechst 33342 stain for visualization of nuclei, and the Click-iT® TUNEL AlexaFluor® Imaging assay were purchased from Invitrogen (Carlsbad, CA). The rabbit monoclonal antibodies against cleaved caspase 8 and GAPDH were both purchased from Cell Signaling Technology (Danvers, MA). Mouse monoclonal antibodies against integrin αvβ5 (15 F11), integrin β3 (LM609), integrin αv (MAB1980), α5β1, and the monoclonal blocking antibody against integrin αvβ5 (P1F6) were purchased from Millipore (Billerica, MA). The purified rat anti-mouse CD29 (clone 9EG7) which recognizes the ligand bound conformation of the β1 integrin  was purchased from BD Biosciences. The rabbit polyclonal antibody to vitronectin, AC7, has been previously described . Pre-immune normal rabbit IgG was used as a control. Alexafluor488-conjugated secondary anti-mouse IgG (H + L) antibody was purchased from Molecular Probes (Eugene, OR). Horseradish peroxidase conjugated secondary antibodies to mouse IgG (H + L) and rabbit IgG (H + L) were purchased from BioRad (Berkeley, CA).
Cell culture, treatment and lysis
The human tumor cell line NCI-H460 was purchased from the American Type Culture Collection (Manassas, VA). NCI-H460 cells were grown in monolayer culture in complete medium (RPMI 1640 with streptomycin-penicillin and glutamax supplemented with 10 % FBS) at 37 °C in a humidified atmosphere containing 5 % CO2. Prior to treatment, cells were serum-starved in RPMI-1640 with 0.1 % BSA for 2 h. For the collection of whole cell lysates, monolayers were washed twice in ice-cold PBS and lysed in whole cell lysis buffer (100 mM Tris–HCl, pH 6.8, 2 % SDS, 10 % glycerol, 100 mM DTT).
Terminal dexoynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay for apoptosis
NCI-H460 cells were cultured in complete medium 48 h or until cells reached ~85 % confluency, rinsed once with PBS and serum-starved in RPMI-1640 with 1 % BSA for 2 h at 37 °C in a humidified atmosphere containing 5 % CO2. Cells were stimulated with recombinant human TRAIL/TNFSF10 (TRAIL) (R&D systems) or PBS as a control treatment as indicated in the figure legends. Apoptosis was assessed by cleaved caspase 8 protein levels via western blot analysis or by TUNEL assay. The TUNEL assay was performed using Click-It® TUNEL AlexaFluor488® Imaging Kit (Invitrogen) according to the manufacturer’s protocol. In brief, cells were fixed with 4 % paraformaldehyde in PBS at room temperature for 20 min and permeabilized with Triton X-100 (0.25 % in PBS) for an additional 15 min. The cells were then washed twice and incubated with 50 μL of terminal deoxynucleotidyl transferase reaction buffer (Component A) for 10 min at room temperature. The buffer was removed and the cells were incubated with TUNEL reaction mixture containing terminal deoxynucleotidyl transferase for 1 h in a humidified chamber at 37 °C for 1 h. Post treatment, cells were washed three times with 3 % BSA in PBS for 2 min each and then incubated with 50 μL of Click-iT reaction mixture (containing Alexa 488) for 30 min at room temperature, protected from light. The cells were then washed with 3%BSA in PBS and the nuclei were stained with Hoechst 33342 for 1 min at room temperature, protected from light. The coverslips were washed twice with PBS before mounting onto a slide with ProLong® Gold Antifade Mount (Life technologies). Cell monolayers were examined using an Olympus BMX-60 microscope equipped with a cooled CCD sensi-camera (Cooke, Auburn Hills, MI), and images were acquired using Slidebook software (Intelligent Imaging Innovation, Denver, CO). Fluorescence images were processed with ImageJ analysis software for the quantification of TUNEL-positive nuclei and total number of nuclei. The number of TUNEL-positive cells in three random (20×) fields was counted and divided by the total number of nuclei to determine the percentage of TUNEL-positive nuclei.
Western blot analysis
Whole cell lysates were collected from treated cells and subjected to SDS-PAGE under reducing conditions and transferred onto nitrocellulose membranes (Schleicher and Schuell Bioscience, Keene, NH). Membranes were blocked for either 2 h at room temperature or overnight at 4 °C with 5 % BSA (w/v) in Tris-buffered saline containing 0.1 % Tween-20 then incubated with primary antibodies for 2 h at room temperature or overnight at 4 °C. Blots were washed with Tween-20 and incubated with horseradish peroxidase-linked secondary antibodies (1:10,000) for 1 h at room temperature. Immunoreactive bands were detected using ClarityTM Western ECL substrate (BioRad). Blots were reprobed after stripping in 62.5 mM Tris–HCl, pH 6.8, 2 % SDS and 10 mM β-mercaptoethanol for 20 min at 60 °C. Western blots were quantified either by ImageJ analysis software (for blots developed on film) or by ChemiDoc™ MP Imaging System with Image Lab (BioRad).
Adhesion assay for integrin activation
Polystyrene non-tissue culture-treated 48-well plates (Greiner bio-one; Monroe, NC) were coated with vitronectin in PBS overnight at 4 °C. Wells were blocked with 3 % BSA in PBS for 2 h in room temperature. Adherent H460 cells were lifted with Cellstripper® (Cellgro) and resuspended in RPMI-1640 containing 0.1 % BSA. Suspended cells (~105) were serum-starved for 1 h then treated with FnIII-1c or PBS, as a control, for an hour at 37 °C. When pharmacological inhibitors to PI3K (Wortmannin, LY294002) and Akt1/2 (VIII) were used, cells were pre-treated with inhibitors for 30 min prior to treatment with FnIII-1c for an additional hour. Treated cells were seeded onto vitronectin-coated wells at a density of approximately 4x104cells/wells and allowed to adhere for 1 h at 37 °C. Adherent cells were quantified by staining with 0.05 % toluidine blue for 1 h. Each well was washed four times and dye was extracted with 10 % acetic acid. Absorbance was measured at 650 nm and corrected for light scattering by subtracting the absorbance at 405 nm.
Fluorescence microscopy for integrin expression
NCI-H460 cells (~104 cells/mL) were cultured in complete medium for 48 h on glass coverslips, then serum-starved for 1 h. Serum-starved cells were washed once with PBS, fixed for 20 min in 4 % paraformaldehyde, permeabilized in 0.5 % TritonX-100 for 10 min, blocked in 1 % BSA and immunostained with monoclonal antibodies against integrins α5β1 (1:200 dilution), αvβ5 (1:200) αvβ3 (1:100) and 9EG7, a monoclonal antibody that detects ligated β1 integrins (1:100) for 1 h at room temperature or overnight at 4 °C. Slides were then incubated with Alexafluor488-conjugated secondary anti-mouse IgG (H + L) antibody for an additional hour at room temperature. Nuclei were visualized with Hoechst 33342 dye. After staining, slides were mounted with Prolong Antifade according to the manufacturer’s instructions (Molecular Probes) and examined using an Olympus BMX-60 microscope equipped with a cooled CCD sensi-camera (Cooke, Auburn Hills, MI). Images were acquired using Slidebook software (Intelligent Imaging Innovation, Denver, CO).
Tissue section staining
Non-small cell human lung carcinoma tissue LC241 NSCLC microarray panels (US Biomax Inc., Rockville, MD), were immunostained using the peroxidase-based ABC system (Vector Laboratories, Burlingame, CA). Vitronectin was detected using a polyclonal antibody to vitronectin and for negative controls, the primary antibody was replaced with pre-immune normal rabbit IgG. Color was developed by reaction with 3,3’-Diaminobenzidine. Tissue sections were counterstained with hematoxylin.
Data are presented as the mean ± SE of at least three independent experiments. Adhesion and TUNEL assay results were analyzed using either a one-way or two-way Anova with Tukey’s post-hoc analysis. Statistical analysis was performed with GraphPad Prism 6 with p < 0.05 considered significant.
FnIII-1c desensitizes NCI-H460 cells to TRAIL-induced apoptosis
FnIII-1c enhances the PI3K-Akt-dependent activation of the αvβ5 integrin
To test whether FnIII-1c was affecting the activation state of integrin αvβ5, we evaluated cell adhesion over a range of vitronectin concentrations. FnIII-1c or PBS control treated cells were seeded onto vitronectin-coated plates and allowed to adhere for 1 h. As displayed in panel B, FnIII-1c significantly increased cell adhesion to vitronectin across a range of coating concentrations, consistent with an increase in integrin activation (Fig. 3b). Experiments were then done to determine whether the change in integrin activation might be required for FnIII-1c’s inhibition of TRAIL-induced apoptosis. Treatment of cells with a blocking antibody to αvβ5 (P1F6) partially restored TRAIL-induced caspase 8 cleavage in FnIII-1c treated cells (Fig. 3c). Analysis of data from several experiments indicated that this restoration was statistically significant (Fig. 3d). Control experiments using LM609, the integrin αvβ3 blocking antibody, or a non-immune mouse IgG, had no effect on FnIII-1c-mediated inhibition of TRAIL-induced caspase 8 cleavage (Fig. 3c–d). These data suggest that the increase in activation and ligation of integrin αvβ5 was contributing to FnIII-1c-mediated inhibition of TRAIL-induced apoptosis.
Inhibition of the PI3K-Akt pathway restores TRAIL-induced apoptosis in FnIII-1c-treated NCI-H460 cells
Human NSCLC tumors express vitronectin
Changes in tissue mechanical properties is a hallmark of solid tumors. Lung cancer is often seen in association with pulmonary diseases characterized by increased tissue rigidity secondary to fibrosis, inflammation and extracellular matrix remodeling. Fibronectin is under complex mechanical regulation and the impact of this regulation on progression of solid tumors is not well understood (reviewed in ). In the present study, we define a molecular mechanism by which unfolding of the first Type III domain of fibronectin may protect NSCLC cells from TRAIL-induced apoptosis. The first Type III domain of fibronectin has been shown to unfold in vitro to support fibronectin polymerization [17, 34, 35] and in vivo to regulate skeletal muscle contraction . Treatment of NCI-H460 cells with the FnIII-1c peptide derived from the first type III domain of fibronectin resulted in the inhibition of TRAIL-induced apoptosis. Pre-incubation of FnIII-1c treated cells with PI3K or Akt inhibitors was sufficient to restore TRAIL-induced cell death, indicating that the PI3K-Akt pathway was required for FnIII-1c-mediated inhibition of TRAIL-induced apoptosis. We also found that FnIII-1c caused Akt dependent activation of the αvβ5 integrin which was required for FnIII-1c’s inhibition of TRAIL signaling. Consistent with this finding an earlier study in the TRAIL-resistant NSCLC cell line, A549, reported that compared to wildtype TRAIL, RGD-TRAIL was more cytotoxic. The investigators concluded that the tumoricidal effect of RGD-TRAIL was due to the interaction of the RGD sequence with integrins αvβ3 and αvβ5 . Altogether the data demonstrate that the signaling pathways activated in response to fibronectin Type III domain unfolding may contribute to Trail-resistance.
The stromal matrix of solid tumors is in a constant state of remodeling where changes in the balance of mechanical forces can alter the topographical display of bioactive sites [38, 39]. The Type III domains of fibronectin are mechanically labile and have been shown to unfold in response to increased cellular contractile forces generated in rigid tissues [40, 41]. As tumor tissue is known to be more rigid than the neighboring normal tissue, tumor stroma should be enriched in unfolded Type III domains. In agreement with this, recent studies have identified unfolded Type III domains in the stromal fibronectin present in breast tumors [42, 43], where the subsequent change in topography of the fibronectin matrix causes an integrin “switch” to promote angiogenesis .
The mechanisms by which Akt protects cancer cells from apoptosis are varied. In the context of TRAIL signaling, Akt has been shown to inhibit cell death by upregulating c-FLIP expression which competes with caspase 8 for recruitment to FADD thereby preventing proper DISC formation [44, 45]. In our study, FnIII-1c had no effect on cFLIP levels (unpublished observations). Instead FnIII-1c stimulated Akt activation and enhanced cell adhesion to vitronectin. This increased adhesion was attenuated by PI3K/Akt inhibitors consistent with FnIII-1c inducing an Akt mediated “inside-out” activation of the αvβ5 integrin. Earlier studies have documented Akt regulation of the activation state of the α5β1, αvβ3 and αIIβ3 integrins [46–48]. Our study is the first to link Akt to αvβ5 integrin activation and suggests that control of integrin activation by Akt may be context dependent. Integrin binding to extracellular matrix is known to promote survival and protect tumor cells against cell death. In many instances, the FAK/Src/PI3K/Akt signaling axis activated by integrin ligation inhibits apoptosis by regulating the expression of anti-apoptotic proteins and cell cycle regulatory genes to prevent both intrinsic and extrinsic cell death (reviewed in ). Very recent studies have shown that fibronectin can overcome the effects of several chemotoxic drugs by mechanisms linked to the activation of Akt [46, 50–53]. In our study, Akt activation preceded integrin activation and prevented the cleavage of caspase 8 consistent with FnIII-1c inhibiting TRAIL signaling by blocking the recruitment of pro-caspase 8 to the DISC.
In addition to their role in the regulation of cell death pathways, caspases can participate in a number of other cellular processes including inflammation, differentiation, proliferation and migration . How these various functional activities of caspases are regulated is not well understood. Caspase 8 is subject to various post-translational modifications such as serine/threonine and tyrosine phosphorylation, ubiquitination and nitrosylation (reviewed in ). One of the non-apoptotic functions of caspase 8 is to promote cell migration by interacting with pathways controlling focal adhesion turnover . Association of caspase 8 with focal adhesion proteins is dependent on cell adhesion , suggesting that increasing the number of ligated integrins may direct caspase 8 to focal adhesions. Therefore, changes in the pattern of ligated integrins may redirect subcellular localization of caspase 8 resulting in the inability of activated death receptors to recruit a critical mass of procaspase to the developing DISC.
In the present study, we localized vitronectin staining in NSCLC tumors to the stroma surrounding blood vessels and to restricted areas of the tumor-stromal interface. These findings are in agreement with a recent study evaluating the expression of vitronectin and the αvβ5 integrin in 215 primary tumors from NSCLC patients. In this study, 70 % of the tumors stained positively for αvβ5 and vitronectin. αvβ5 was localized on the membrane of tumor cells while vitronectin was seen exclusively in the stroma surrounding the blood vessels . In earlier studies, we have shown that the lung stroma adjacent to the border of the infiltrating tumor is heavily stained for fibronectin and smooth muscle actin suggesting that the fibroblasts aligned along the fibronectin matrix are myofibroblasts .
Taken together, these data identify a novel pathway by which changes in the mechanical forces within the stroma can alter the topography of the fibronectin matrix thereby contributing to cancer cell resistance to therapy-mediated cell death. The stretching of fibronectin coupled with the availability of vitronectin in the tumor stroma creates a specialized niche to protect tumor cells from therapies designed to activate extrinsic cell death pathways. Therefore, coupling therapies directed at TRAIL pathways with those controlling mechanical signaling may provide novel approaches for the treatment of TRAIL resistant tumors.
Akt, alpha serine/threonine kinase; DISC, death inducing signaling complex; DR4, death receptor 4; DR5, death receptor 5; ECM, extracellular matrix; FADD, Fas-associated protein with death domain; FLICE, FADD-like interleukin 1 beta concerning enzyme; FnIII fibronectin Type III domain; Fn, fibronectin; NSCLC, non-small cell lung cancer; PGS, progressive free survival; PI3K, phosphatidylinositol-3-kinase; TNF, tumor necrosis factor; TRAIL, tumor necrosis factor related apoptosis inducing ligand
This was supported by NIH CA058626 and by DoD LC090169 to PM-L.
Availability of data and materials
Reagents and materials will be made available upon request.
CC performed all experiments and participated in experimental design, data interpretation and manuscript writing. CH prepared and purified all proteins. DJ read and interpreted stained lung tumor sections. PML was responsible for experimental design, data interpretation and manuscript writing. All authors have read and approved the final manuscript.
The authors declare that they have no competing interests.
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