TGFBI expression is associated with a better response to chemotherapy in NSCLC

Loss of TGFBI expression has been associated with the tumorigenic phenotype of several carcinomas including lung cancer [[20], and Additional file 1]. It has also been correlated with enhanced sensitivity to paclitaxel and etoposide in ovarian carcinoma [22] and a lung adenocarcinoma cell line [20], respectively.

In order to determine if loss of TGFBI expression correlated with the chemotherapeutic response in clinical samples, we retrospectively analyzed pre-chemotherapy TGFBI protein expression in a series of 47 stage IV NSCLC samples by immunohisto-chemistry. TGFBI expression was significantly (p = 0.003) higher in patients who responded to chemotherapy (average TGFBI H score = 170) compared to patients whose cancer progressed (average score = 110) or who had stable disease (average score = 120) (Figure 1). We did not observe any correlation between TGFBI expression and other clinico-pathological parameters analyzed such as gender, age or tumor histology. Detailed information regarding TGFBI expression in each tumor sample, patient outcome and the type of chemotherapy administered is provided in Additional file 2.

We also performed Kaplan-Meier analysis of overall survival on the same series of patients. Data shown in Additional file 3 illustrate that patients expressing higher levels of TGFBI show a trend towards better survival (p = 0.053). This data, in spite of not being significant, suggests its possible use as a candidate prognostic factor. Nevertheless, analyses performed in greater number of samples should be made to definitively test this.

To identify the mechanism underlying the role of TGFBI in tumor cell response to chemotherapy, we transiently over-expressed TGFBI in the metastatic NSCLC cell line NCI-H1299, which has a low basal expression of this protein (H1299-TGFBIve). Additionally, we silenced TGFBI in non-metastatic NSCLC NCI-A549 cells, which express high basal levels of this protein (A549-TGFBIsi). Basal expression and the efficiency of TGFBI over-expression and silencing are shown in Additional file 4. Using this experimental approach, we determined the effects of TGFBI expression on cell viability using the neutral red assay. TGFBI silencing in A549 cells increased cell viability while over-expression in H1299 cells diminished survival (Figure 2).

Since cells that over-expressed TGFBI showed reduced viability, we postulated that they may be more sensitive to chemotherapy than cells not expressing this protein. To explore this, TGFBI over-expressing (H1299-TGFBIve) or silenced (A549-TGFBIsi) NSCLC cells were treated with paclitaxel, etoposide, cisplatin or gemcitabine and measured their IC₅₀. A549-TGFBIsi cells displayed a higher IC₅₀ for all drugs assayed compared to treated non-transfected or empty vector-containing cells, with more prominent effects observed in cells treated with paclitaxel and etoposide (Figure 3A). On the contrary, H1299-TGFBIve cells displayed a significant decrease in their IC₅₀ (Figure 3B) compared to control cells. We measured PARP cleavage in A549-TGFBIsi and H1299-TGFBIve cells by Western blot analysis after their exposure to etoposide. After etoposide treatment, PARP cleavage in A549-TGFBIsi cells was reduced (Figure 3C), whereas H1299-TGFBIve displayed higher PARP activation (Figure 3D). Therefore, we confirmed that TGFBI expression augmented cell death in NSCLC cells after exposure to chemotherapeutic drugs. Interestingly, the increase observed in cell death following chemotherapy occurred independently of the drug used and, therefore, of the cellular process targeted by these compounds (cytoskeleton remodeling and cell proliferation), which suggests that elevated expression of TGFBI has a direct pro-apoptotic effect on NSCLC cells. To test these, we measured cell viability in non treated NSCLC cells in which TGFBI had been silenced (A549 NSCLC cells) or over-expressed (H1299 NSCLC cells). The results obtained confirmed our hypothesis: those cells that had TGFBI silenced were more viable, while on the contrary, if this protein was over-expressed they presented reduced cell viability (Additional file 5).

Given that TGFBI is an extracellular protein, we next sought to ascertain if the observed effects were reproducible by the addition of exogenous recombinant TGFBI (rh-TGFBI). We measured caspase 3/7 activity in A549 and H1299 NSCLC cells after being exposed to increasing amounts of rh-TGFBI. As shown in Figure 4A, no pro-apoptotic effect was observed in response to low concentrations of recombinant TGFBI but, following exposure to 20 μg/mL rh-TGFBI, a 1.4- and 1.7-fold increase in caspase 3/7 activity was seen in A549 and H1299 cells, respectively. We also observed the same results when the equivalent amounts of recombinant protein were used to coat 96-well plates before seeding the carcinoma cells onto them (Figure 4B).

To determine if NSCLC binding to soluble recombinant TGFBI also contributed to improved responses to chemotherapy, we analyzed caspase 3/7 activation in NSCLC cells exposed to 20 μg/mL rh-TGFBI and treated with etoposide at a concentration of 5 μM and 50 μM for A549 cells and H1299 cells, respectively. NSCLC cells treated with etoposide in the presence of recombinant TGFBI displayed higher caspase 3/7 activity compared to cells treated with the chemotherapeutic drug alone (Figure 4C). As expected, when seeding A549 and H1299 cells onto TGFBI coated 96-well plates, an increased response to etoposide was also induced (Figure 4D).

Once we demonstrated that TGFBI induced apoptosis in our system, we next wanted to ascertain which cell surface receptor was required. Integrins are the only type of cellular receptors that have been shown to mediate TGFBI signaling. To determine which integrin was involved in TGFBI binding to NSCLC cells, we performed cell adhesion assays using NSCLC cells cultured onto plate-bound rh-TGFBI (1 μg/mL) in the presence of a variety of anti-integrin blocking antibodies. The adhesion of A549 and H1299 cells to rh-TGFBI was blocked only in the presence of the anti-αvβ3 integrin monoclonal antibody (Figures. 5A and 5B). A noticeable difference in basal cell adhesion to rh-TGFBI was observed between A549 and H1299 cells, the former showing lower adhesion than the latter. When we determined by flow cytometry the expression levels of the integrin αvβ3 on the surface of both cell lines, we observed that A549 cells expressed lower αvβ3 integrin than H1299 cells. This is probably the cause of their lower adhesion to TGFBI coated plates (Figure 5C).

It has been previously shown that αvβ3 integrin mediates paclitaxel sensitivity of ovarian cancer cells that transiently expressed TGFBI [22]. Therefore, to determine if this integrin influences TGFBI-mediated NSCLC cell susceptibility to chemotherapy, we incubated NSCLC cells with a blocking antibody specific for αvβ3, 1 h before the addition of rh-TGFBI (20 μg/mL) for 24 h and an further exposure to etoposide (5 μM for A549 cells and 50 μM for H1299 cells) for additional 24 h. As shown in figure 5D, blocking αvβ3 integrin resulted in impaired TGFBI-induced caspase 3/7 activity in both the presence and absence of etoposide. All these results demonstrated that this integrin participates in TGFBI-mediated induction of NSCLC cell apoptosis.

TGFBI contains a carboxy-terminal Arg-Gly-Asp (RGD) integrin binding sequence. Given that integrin-mediated binding of a cell to the ECM or to small soluble peptides can have contrasting effects on survival, with the latter resulting in survival and the former leading to cell death [23], we addressed whether TGFBI-derived RGD-containing peptides played a role in TGFBI-mediated apoptosis.

The supernatants from the culture of A549 and H1299 NSCLC cells transiently transfected with the TGFBI expression vector were collected 24 h after transfection, and filtered through a membrane with 3 kDa molecular weight cut-off. Two different cell supernatants were obtained (TGFBI < 3 kDa and TGFBI > 3 kDa). They were separately added to A549 and H1299 cell cultures and, 24 h later, caspase 3/7 cleavage was evaluated to determine if TGFBI-derived peptides influenced cell apoptosis. NSCLC cells over-expressing TGFBI generated a TGFBI-specific band, which was smaller than the full-length 68 kDa recombinant protein, indicating that the TGFBI protein was digested (Figure 6A). The results presented in figure 6B demonstrated that only the smaller TGFBI-derived fragments (TGFBI < 3 kDa) induced caspase 3/7 activity in both A549 and H1299 NSCLC cells, while supernatants containing proteins larger than 3 kDa in size, or those obtained from non-transfected cells, had no effect on caspase activity. Even more, cell supernatants containing proteins larger than 3 kDa were unable to enhance the etoposide-induced increase in caspase 3/7 activation, whereas those supernatants containing smaller TGFBI fragments increased caspase activation similar to that induced by treatment of NSCLC cells with recombinant TGFBI protein. To support these observations, we exposed H1299 cells to short synthetic peptides derived from the RGD region of the TGFBI protein described by Kim and co-workers [24]. Results shown in Additional file 6 demonstrated that peptides containing the full RGD sequence induced cell death while a RGE mutant did not induce it.

Additionally, we found that the pro-apoptotic effects of the TGFBI < 3 kDa supernatants were abrogated by pre-incubating the NSCLC cells with an anti-αvβ3 integrin blocking antibody. We further confirmed the role of the αvβ3 integrin in TGFBI induced cell death by transfection of H1299 with an anti-β3 integrin sh-RNA plasmid and further exposing these cells to 3 < kDa supernatants derived from TGFBI over-expressing cells for 48 h. Caspase 3/7 activity remained basal in β3 integrin silenced cells whereas it was induced in H1299 cells transfected with a scramble-shRNA (Additional file 7).

It has been previously demonstrated that caspase-8 is recruited to a complex containing unligated αvβ3 integrins, where it is activated and mediates apoptosis [25]. In order to determine whether this caspase pathway is activated by TGFBI fragments, we measured caspase 8 activation in NSCLC cells exposed to supernatants containing TGFBI fragments < 3 kDa. Caspase-3/7 activation peaked 24 h after exposure to cell supernatants, while caspase-8 activation peaked 12 h earlier, which was expected due to its known previous activation in the caspase activation cascade (Figure 6C and 6D).

All of these results demonstrated that proteolytic fragments derived from TGFBI induced cell death through binding to the αvβ3 integrin on the surface of NSCLC cells and the subsequent activation of caspases 8 and caspase 3/7 signaling. These findings may explain the important association observed between elevated TGFBI expression and the response to chemotherapy found in NSCLC clinical biopsies.

To analyze NSCLC susceptibility to chemotherapy, a series of retrospective samples from 47 stage IV NSCLC patients were obtained prior to chemotherapy at the Clínica Universidad de Navarra between 2000 and 2006. Eligible patients needed to be 18 or older at the time of treatment, with a histological confirmed diagnosis of stage IV NSCLC, treated in our institution with a platinum-taxane combination regimen in a first-line setting and followed at least until tumor response assessment. In addition, sufficient tumor sample from original biopsy was required in order to perform immunohistochemical analysis. The records of 103 stage-IV NSCLC consecutive patients treated in our institution were initially selected. After evaluation, 22 of the 103 patients were excluded because chemotherapy was not administered in a first-line setting. Other 19 patients were not eligible due to unavailability of sufficient tumor sample to perform immunohistochemical analysis. Finally, 15 patients initially treated in our institution were lost during follow-up before tumor response was assessed and thus were also excluded. Therefore the final number of samples analyzed was reduced to 47 cases.

Clinico-pathological features of these 47 patients are shown in Additional file 2. Histological diagnosis was performed according to the WHO classification [41]. REMARK criteria were followed throughout all the study [42]. Criteria for response were established as already published following RECIST criteria [43]. Complete response was defined as the disappearance of all target and non-target lesions, and the normalization of tumor marker level confirmed by repeat assessments that should be performed no less than 4 weeks after the criteria for response is first met. During this time no new lesions should appear. Partial response was defined as a greater than or equal to 30% decrease in the sum of the largest one-dimensional measurements, maintained for a minimum of 4 weeks. Stable disease was defined as change in the sum of the products or diameters, respectively, insufficient for partial response and progressive disease, maintained for a minimum of 4 weeks from baseline [44]. The study protocol was approved by the ethics committee.

Formalin-fixed paraffin-embedded tissue sections were evaluated. Endogenous peroxidase activity was quenched and antigen retrieval was carried out by pressure cooking. Non-specific binding was blocked using 5% normal goat serum in Tris-buffered saline (TBS) for 30 min. Sections were incubated with anti-TGFBI antibody (1:25; Proteintech group, Chicago, USA) overnight at 4°C. Sections were then incubated with Envision polymer (Dako, Glostrup, Denmark) for 30 min at room temperature. Peroxidase activity was developed using diaminobenzidine and counterstained with hematoxylin before mounting in DPX mounting medium (BDH Chemical, Poole, UK). The specificity of the antibody was assessed by Western blot analysis of proteins from lung tumor sections (Additional file 1). Negative controls were carried out by omission of the primary antibody or incubation with an isotype control antibody.

Two independent, blinded observers (M.J.P and M.I.) evaluated the intensity and extensiveness of staining in all of the study samples. The evaluation of TGFBI expression was performed using the H-score system [45]. Briefly, the percent of positive cells (0-100%) was scored and the intensity of staining was assessed in comparison to an external positive control (1⁺, mild; 2⁺, moderate and 3⁺, intense labeling). A final H score (0-300) was established by multiplying the percentage of labeled cells and the intensity of staining. Disagreements were resolved by common re-evaluation.

Human NSCLC cell lines derived from adenocarcinoma (NCI-A549) and large cell carcinoma (NCI-H1299) were purchased from American Type Culture Collection (ATCC, LGC-Promochem SL, Barcelona, Spain) and grown in RPMI 1640 medium (GIBCO, Barcelona, Spain) supplemented with 10% FBS and penicillin-streptomycin at 100 U/mL each. Cell cultures were incubated at 37°C in a humidified 5% CO₂ incubator.

To over-express TGFBI in H1299 cells (H1299 TGFBIve), 2 × 10⁵ cells/well were transfected with 1 μg of the pCMV6-XL4 plasmid vector containing TGFBI cDNA (Origene, MD, USA) using 2 or 3 μl of FuGENE 6 Transfection Reagent (Roche, IN, USA) in 97 μl of Opti-MEM medium (Invitrogen, Barcelona, Spain) following manufacturer's instructions.

To silence TGFBI expression, 1 × 10⁶ A549 cells/mL (A549-TGFBIsi) were transfected by electroporation with 10 μg of a commercially available shRNA targeting TGFBI cloned in a pRS plasmid vector (Origene, MD, USA). Cell transfections were performed in Opti-MEM medium (Invitrogen) using a Biorad Gene Pulsar I electroporator (Hercules CA, USA), with the capacitance set at 100 F and the voltage at 500 V. Transfection efficiency was confirmed by Western blot of NSCLC supernatants (Additional file 4).

For cell viability assays, 10⁴ NSCLC cells were plated in 96-well plates (TPP, St. Louis MO, USA) in complete RPMI medium as described [46]. To measure the role of TGFBI expression in NSCLC sensitivity to chemotherapy, A549 TGFBIsi and H1299 TGFBIve cells were seeded in 96-well plates. When cells reached confluence, increasing amounts of the four different chemotherapeutic drugs were added to NSCLC cells in complete cell culture media for 48 h and cell viability was assayed. The IC₅₀ for etoposide in non-transfected cells was determined experimentally and used to measure caspase 3/7 activity and PARP cleavage (50 μM for H1299 cells and 5 μM for A549 cells).

To determine the influence of rh-TGFBI protein on cell viability, soluble recombinant human TGFBI (rh-TGFBI) purchased from R&D, was added to the cells in increasing concentrations (0.1, 1, 5, 10 and 20 μg/mL) and cell viability was assayed after 24 h.

To determine which integrin is involved in NSCLC cell adhesion to TGFBI, 1 × 10⁶ cells/mL were labeled with 10 μM calcein-AM (Fluka, Steinheim, Germany) in adhesion-medium (RPMI 0.5% BSA, 20 mM HEPES). Cells in suspension were incubated with gentle rocking for an hour in the presence 1 μg/mL of each monoclonal anti-integrin blocking antibody. Afterwards, 10⁴ cells were seeded onto 1 μg/mL rh-TGFBI coated wells, and cell adhesion was assayed as described [46]. Six replicates were plated for each condition. The following blocking monoclonal antibodies were used: anti-α₁ integrin (MAB1973Z, Chemicon International, CA, USA), anti-β₃ integrin (MAB2023Z, Chemicon International), anti-α₄ integrin (340976, Becton Dickinson, NJ, USA), anti-integrins αv, β₁, β₂, α₅β₁, αvβ₃, αvβ₅ (Integrin Classics Kit, ECM435, Chemicon Interational) and a nonspecific IgG (MAB004, R&D Systems).

To determine the effect of TGFBI expression on PARP cleavage in the NSCLC cells in response to chemotherapy, 10⁶ NSCLC cells were cultured in RPMI complete medium on 10 cm² plates and allowed to adhere for 24 h. After this time, etoposide was added at the IC₅₀ (50 μM for H1299 cells and 5 μM for A549 cells) for 48 h. Then, protein extracts were collected and transferred to nitrocellulose membranes as described [46]. The membranes were blocked by incubating them overnight in a solution of 5% non-fat dry milk in PBS-Tween. Primary PARP antibody (Cell Signaling, MA, USA) incubation was performed for 1 h at room temperature (1:1000 dilution). Membranes were washed three times with PBS-Tween 0.2% and incubated with an HRP-conjugated anti-rabbit IgG (sc-2030, Santa Cruz Biotechnology, CA, USA) in 5% non-fat dry milk in PBS-Tween. The signal was developed using the Lumi-light^PLUS Western Blotting Kit (Roche Molecular Biochemicals, Mannheim, Germany).

For determination of integrin αvβ3 expression on the surface of NSCLC cells, 1 × 10⁵ cells A549 and H1299 cells were cultured to subconfluency in 6 well plates containing 1 mL of complete cell growth media. Afterwards, the cells were harvested and resuspended in 100 μl of staining buffer (5% FBS, EDTA 2 mM in PBS) and incubated for 10 min at room temperature with a concentration of 1 μg/ml of the, anti-integrin αvβ3 (Integrin Classics Kit, ECM435, Chemicon Interational) monoclonal antibody or a non-specific IgG (MAB004, R&D Systems). Afterwards, the cells were washed with staining buffer and subsequently incubated with 1:500 green-fluorescent Alexa Fluor 488 anti-mouse-secondary antibodies (Molecular Probes) at room temperature for 10 min. Cells were washed twice with staining buffer and analyzed on a FACScan flow cytometer using Cellquest software (BD-PharMingen).

To determine caspase 3/7 activity, 10⁴ cells were cultured in RPMI complete medium on 96-well plates coated with increasing amounts of rh-TGFBI (rhTGFBI) and allowed to adhere for 24 h. After this time caspase 3/7 activity was measured. In some instances, the cells were allowed to adhere to non-coated wells for 8 h and then incubated with increasing amounts of rh-TGFBI for 24 h. To measure caspase 3/7 activity after etoposide treatment, NSCLC cells were incubated with this drug at the IC₅₀ (50 μM for H1299 cells and 5 μM for A549 cells) for additional 24 h after being treated with or seeded onto rh-TGFBI, and caspase activity was measured afterwards.

For αvβ3 integrin blocking experiments, 10⁴ cells were cultured in RPMI complete medium on 96-well plates and always allowed to adhere to non-coated surfaces for 8 h. After this time, the cells were pre-incubated for 1 h with 1 μg/mL of the monoclonal anti-αvβ3 integrin antibody (Chemicon, IL, USA). Then, 20 μg/mL of rh-TGFBI was added to the cells without removing the anti-integrin monoclonal antibody. When indicated, 24 h latter, etoposide was added at its IC₅₀ for additional 24 h. Caspase 3/7 activity was estimated using the Caspase-Glo 3/7 Assay (Promega, Madrid, Spain) and quantified on a Polar Star Galaxy plate reader (BMG, Offenburg, Germany), using 530 emission filters. Three replicates were assayed for each condition.

To analyze the effects of RGD peptides on the apoptosis of NSCLC cells, we collected supernatants from A549 and H1299 cultures after cells were transiently transfected with the TGFBI expression vector. The amount of TGFBI protein in the cell supernatants was estimated by densitometric analysis of Western-blot bands using as reference, the intensity of 10 ng of recombinant protein loaded in the first lane of each gel. NSCLC cell supernatants were collected 24 h later and filtered through a 3 kDa exclusion column (Centricon, MA, USA). Both the concentrate (TGFBI < 3 kDa) and the filtrate (TGFBI > 3 kDa) were added to different A549 and H1299 cultures at a final concentration of 1× for 4, 8, 12 or 24 h. At each time-point, caspase 3/7 and caspase 8 activity were measured using the Caspase-Glo Assay (Promega) as above.

Figure legends for additional figures and additional Material and Methods are provided as additional files 8 and 9 respectively.