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. Author manuscript; available in PMC: 2011 Sep 15.
Published in final edited form as: Cancer Res. 2010 Sep 7;70(18):7125–7136. doi: 10.1158/0008-5472.CAN-10-1547

The Neutrophil Elastase Inhibitor, Elafin, Triggers Rb-Mediated Growth Arrest and Caspase-Dependent Apoptosis in Breast Cancer

Joseph A Caruso 1, Kelly K Hunt 1,2, Khandan Keyomarsi 1
PMCID: PMC2940941  NIHMSID: NIHMS227130  PMID: 20823156

Abstract

Elafin, an endogenous inhibitor of neutrophil elastase, is expressed in human mammary epithelial cells, but is transcriptionally downregulated in breast cancer cells. We hypothesized that elafin may exert a tumor suppressive activity in the context of breast cancer. In this study, we demonstrate that the Retinoblastoma (Rb) pathway governs the anti-tumor properties of elafin. In breast cancer cells with functional Rb, the expression of elafin triggered Rb-dependent cell cycle arrest. Elafin also exhibited suppressive activity in breast cancer cell lines lacking Rb, but this was associated with an induction of caspase-3 dependent, p53 independent apoptotic cell death. Normal mammary epithelial cells were not affected by elafin. Collectively, these results argue that elafin mediates tumor suppressive effects that are cytostatic or cytotoxic depending on Rb status. Our findings suggest that elafin could be engineered as a therapeutic modality to treat breast cancer without toxicity to normal proliferating cells.

Keywords: Elafin, Human Neutrophil Elastase, Serine Protease, Breast Cancer, Retinoblastoma

Introduction

The serine protease inhibitor, elafin, was originally purified from skin lesions associated with the inflammatory disease, psoriasis. It was shown to be a specific inhibitor of Human Neutrophil Elastase (HNE) and highly related Proteinase 3 (1). Elafin has a role in inflammation, where it is hypothesized to act as part of the protective barrier erected by epithelial cells against tissue degrading, leukocyte-secreted proteases (2, 3). Elafin may also have a role in cancer progression. Studies have shown that elafin is expressed in well-differentiated squamous cell carcinomas of the skin, head/neck, and esophagus, and is absent in poorly differentiated tumors at these sites; suggesting that elafin downregulation may have a role in the development of a poorly differentiated and aggressive tumor phenotype (4-7). The majority of breast tumor derived cell lines demonstrate transcriptional downregulation of elafin, when compared to human mammary epithelial cells (HMECs) (8). C/EBP β transcriptional elements regulate elafin expression in HMECs, while the frequent deregulation of C/EBP β in breast cancer results in transcriptional loss of elafin expression in tumor cells (9).

In the present study, we hypothesize that elafin possesses novel anti-tumor properties in breast cancer. Our analysis has defined an important role for Rb in the determination of cell fate following elafin expression. In normal HMECs with functional Rb, the overexpression of elafin had no affect; however in tumor cell lines with functional Rb, elafin expression results in a G0/G1 cell cycle arrest. In both normal and tumor derived breast cell lines with loss or inactivation of Rb, elafin expression results in caspase-3 dependent apoptotic cell death.

Approximately one-quarter of all breast tumors exhibit homologous loss of Rb (10, 11). Rb loss is frequently associated with aggressive breast cancer subtypes, including luminal B or basal-like (12, 13). These subtypes of breast cancer are resistant to currently available targeted therapies (i.e. anti-estrogens and anti-HER2) (14, 15). An effective targeted therapy capable of specifically targeting breast tumors with Rb loss may have significant clinical utility in highly aggressive breast cancer subtypes.

Materials and Methods

Cell lines and Culture Conditions

All cell lines were maintained in a humidified tissue culture incubator at 37°C and 6.5% CO2. All tumor derived cell lines (MDA-MB-157, MDA-MB-231, MDA-MB-468, MCF-7, ZR75.1, and T47D) were obtained from ATCC. MDA-MB-468 cells were maintained in DMEM (HyClone) and all other tumor cell lines were maintained in α-MEM (HyClone), both supplemented with 10% fetal calf serum (Atlanta Biological). The immortalized human mammary epithelial cell lines (HMECs) 76NE6, 76NF2V, and 76NE7 were obtained from Dr. V. Band and were maintained in DCFI-1 media (D1-media) (16, 17). The 76NE6 cell line lacks p53, the 76NE7 cell line lacks Rb and other pocket proteins, and the 76NF2V expresses both p53 and Rb. Rb and p53 status of all the cell lines used is indicated in Supplementary Table 1. All cells were cytogenetically tested and authenticated before the cells were frozen. Each vial of frozen cells is thawed and stayed in culture for a maximum of 6 weeks. There were enough frozen vials for each cell line to ensure that all cell based experiments were performed on cells that have been tested and in culture for 6 week or less.

Generation of clonally derived cell lines

MCF-7 cells stably expressing a siRNA vector targeting p53 were a generous gift form Dr. Xinbin Chen. MDA-MB-231 and MCF-7 cells stably expressing shRNA directed against Rb were a generous gift from Dr. E. Knudsen. MCF-7 cells with reconstituted caspase 3 were a generous gift from Dr. B. Fang. MCF-7 RBKD and MCF-7 shRNA control cell lines expressing caspase 3 or pcDNA3.1 empty vector were generated by transfection of the MCF-7 RBKD and MCF-7 shRNA control cell lines with 6 μg of pcDNA3.1/Casp3 (also a gift from Dr. B. Fang) or 6 μg of pcDNA3.1 empty vector using Genejuice reagent (Novagen), according to manufacturer's instructions. Cells expressing these vectors were selected in α-MEM containing 0.5 mg/ml G418 (Mediatech) and 2 ug/mL puromycin (Sigma) for four weeks. Single-cell clones were selected and expanded in culture media supplemented with 0.1 mg/ml G418 and 2 ug/mL puromycin (Sigma) and screened by western blot analysis for the expression for Rb and caspase 3

Adenovirus

Elafin and Firefly Luciferase transgenes were expressed downstream of a cytomegalovirus (CMV) promoter in replication incompetent adenovirus type 5 with deletions of the E1 and E3 genes. The elafin cDNA encodes the 57-C-terminal amino acids containing the elastase inhibitory domain (1). Viruses were amplified in the packaging cell line AD-293 (Stratgene) and purified in a CsCl gradient by centrifugation at 176 000 g (Beckman ultracentrifuge). Multiplicity of infection (MOI) was calculated based on the number of plaque forming units (pfu) in AD-293 cells. For infections 2 × 106 cells were plated on a 100mm plate, after 24 hours cells were washed with sterile PBS and treated with Ad-elafin, Ad-luciferase, or PBS in four mL of media. After two hours of infection the media was removed and replaced with fresh culture media. All tumor cell lines were optimally infected at an MOI of 1 except for MDA-MB-231, which along with HMEC cell lines were infected at an MOI of 2. Transgene expression was determined by western blot analysis. Infection efficiency was examined using an identically titered adenovirus carrying GFP, all cell lines were infected at greater than 90% efficiency.

Drug Treatment

The pan-caspase inhibitor zVad-fmk (calbiochem) was solubilized in DMSO at 50 mM and diluted in media at a concentration of 50μM.

Growth Curves

5000 tumor cells or 2500 HMECs were plated in each well of a 24 well plate. After 24 hours, cells were treated with Ad-Elafin, Ad-Luc, or PBS in triplicate. Cells were harvested every 24 hours for the indicated durations. At each time point cell number was determined using the trypan blue (Fluka) exclusion test.

DNA Content Analysis

Following indicated treatment, 2×106 cells were fixed by suspension in ice-cold 70% ethanol. Cells were stored at 4°C at for least 24 hours, washed with PBS, and stained in 0.5 mL of propidium iodide solution (10 μg/mL PI (Molecular Probes) + 20 μg/mL ribonuclease A (Sigma) in PBS containing 0.5% Tween-20 (Sigma) and 0.5% BSA (Sigma)) overnight at 4°C. The cells were incubated for 30 minutes at 37 °C, PI fluorescence was measured using a BD FACScalibur flow cytometer, and analyzed based on DNA content using FloJo software.

BrdU incorporation

Cells were plated in a 100 mm3 plate and treated 24 hours later with Ad-Elafin, Ad-Luc, or PBS. 48 hours post-treatment, cells were pulsed for 1 hour with 10 μM BrdU (Invitrogen). Cells were prepared and stained with FITC-conjugated anti-BrdU monoclonal antibody (BD Bioscience) according to manufactures instruction. Cells were co-stained with PI solution. FITC and PI fluorescence was measured using a BD FACScalibur flow cytometer and analyzed using FloJo software.

TUNEL Assay

Both the floating and adherent cells were harvested 72 hours post treatment and submitted to the TUNEL assay (APO-BrdU TUNEL Assay Kit; Invitrogen). The assay was preformed according to the manufacturer's instructions. The included Alexa-Fluor 488 conjugated anti-BrdU antibody was used in all cases except where GFP expression from stably transfected plasmids (RBKD and control cell line) interfered with detection of the Alexa-Fluor 488 dye; in this case Alexa-Fluor 647 conjugated Anti-BrdU antibody (BD) was substituted.

Western blot analysis and Immunopercipitation/Kinase Assay

Cells lysates were prepared and subjected to western blot analysis as previously reported(18). For each western blot 50 μg onto a 7.5% (Rb, phospho-Rb, and PARP) or 13% (Caspase 3, cleaved caspase 3, elafin, and actin) SDS-PAGE gel. Primary antibodies used were mouse monoclonal elafin (HM2063; HyCult Biotechnology), mouse monoclonal Rb 4H1 (Cell Signaling Technology), rabbit polyclonal phospho-Rb serine 807/811 (Cell Signaling Technology), rabbit polyclonal phospho-Rb serine 780 (Cell Signaling Techonology), rabbit polyclonal PARP (Cell Signaling Technology), rabbit polyclonal Caspase 3 (Cell Signaling Technology), rabbit polyclonal Cleaved Caspase 3 Asp 175 (Cell Signaling Technology), and mouse monoclonal Actin (Chemicon International, Inc.).

In vitro CDK4 kinase assay was preformed as previously described (19)using rabbit polyclonal CDK4 antibody (Santa Cruz Biotechnology) and GST-Rb 769 substrate (Santa Cruz).

Cell Viability Assay

Cell viability was measured by using the MTT assay in a 96-well plate format. Briefly, 5000 cells were plated and treated as indicated, then incubated in 50 μl of 2.5 mg/mL MTT in serum-free media for 4 hours, solubilized (20mL 1N HCL, 50 mL 10% SDS, 430 mL isopropyl alcohol) for one hour on a horizontal shaker, and quantified using a spectrophotometer (Victor3, Perkin-Elmer) at a wavelength of 590 nm.

Statistical analysis

All pairwise comparisons were analyzed using the student t-test. All error bars represent the Standard Error of the Mean (SEM). Many of the error bars on the growth curves are obscured by the size of the symbol marking each data-point; in these cases the SEM associated with these measurement is negligible when compared to the large range on the y-axis, this is especially true for early timepoints when cell number is exceptionally low.

Results

Exogenously expressed elafin induces apoptosis in HMECs lacking functional Rb

Elafin is cell cycle regulated with the lowest relative expression occurring during the S-phase of the cell cycle, indicating a possible role for elafin in cell cycle progression(8). . Therefore we first investigated if the exogenously expressed elafin can affect growth of actively proliferating HMECs. For these experiments we overexpressed elafin using an adenovirus system; sub-confluent 76NE6, 76NF2V, and 76NE7 cells were treated with adenoviral elafin (E), adenoviral luciferase (L), or PBS (P). Lysates were subjected to western blot analysis to demonstrate expression of the elafin transgene in comparison to endogenous elafin expression in growth factor deprived (the highest relative expression occurring in G0/G1; unpublished data J.Caruso) 76NE6 cells (Fig 1A-compare lane C, to lane E for each cell line). The overexpression of elafin causes no statistically significant change in the doubling times of Rb proficient cell lines 76NE6 (PBS:16±1.1 hours, luciferase:15±0.5 hours, and elafin 15±0.3 hours) and 76NF2V(PBS:18±0.6 hours, luciferase:18±0.6 hours, and elafin 20.3±0.5 hours). However, overexpression of elafin in 76NE7 cells, which are devoid of Rb, resulted in a significant decrease in cell number by 96 hours post-treatment (Fig 1A). In order to determine if the decrease in cell number seen in 76NE7 cells is due to an Ad-elafin induced block in proliferation or induction of apoptosis we analyzed BrdU incorporation into DNA (Fig 1B) and measured percentage of TUNEL positive cells (Fig 1C). There was no statistically significant change in BrdU incorporation in any of the cell lines tested, 76NE6, 76NF2V, or 76NE7, upon ectopic expression of elafin (Figure 1B). However, when we measured DNA fragmentation using the TUNEL assay, we found that there was a significant increase in TUNEL positive cells in Ad-elafin treated 76NE7 cells compared to Ad-luciferase treated 76NE7 or Ad-elafin treated 76NE6 or 76NF2V cells (Figure 1 C). These results demonstrate that treatment of 76NE7 cells with Ad-elafin results in an increase in apoptosis leading to a significant reduction in cell number 96 hours post treatment. These results also suggest that elafin induces apoptosis preferentially in Rb deficient cells.

Figure 1. Exogenously expressed elafin induces apoptosis in HMECs lacking functional Rb.

Figure 1

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76NE6, 76NF2V, and 76NE7 cells were treated with PBS (P), adenoviral luciferase (L), or adenoviral elafin (E). (A) Cells were harvested 48 hrs post-treatment and subjected to western blot analysis with indicated antibodies. 76NE6 cells cultured in D3-media (no growth factors) were prepared as a control (C) for elafin expression. Relative elafin levels were calculated by densitometry, displayed above. (B) Growth was monitored by trypan blue exclusion test every 24 hours for 96 hours. S . 76NE7 cells overexpressing elafin showed a statistically significant difference (t-test), in cell number at 72 hours (vs. PBS p=0.0122 and vs. Ad-luc p=0.0125) and 96 hours (vs. PBS p<0.0001 and vs. Ad-luc p<0.0001). (C) Proliferation was assessed 48 hours post-treatment by measuring BrdU incorporation; repeated in triplicate. (D) Apoptosis was assessed 72 hours post-treatment by TUNEL assay. All experiments were repeated in triplicate.

Expression of elafin in Rb negative breast cancer cells results in apoptosis

Based on the finding that elafin induces apoptosis in Rb deficient HMECs, we next asked if Rb deficiency in breast cancer cells causes a similar susceptibility to elafin induced apoptosis. For these studies we examined the role of elafin in three breast cancer cell lines with functional/wild-type Rb (MCF-7, ZR75.1, and T47D) and three cell lines deficient in Rb (MDA-MB-157, MDA-MB-468 and MDA-MB-436). All six cell lines were treated with adenoviral elafin (E), adenoviral luciferase (L), or PBS (P) and were examined for growth kinetics, BrdU incorporation and TUNEL postivity (Figure 2). Western blot analysis showed that adenoviral elafin resulted in 1-3 fold elafin expression compared to control, (76NE6 cells growth factor deprived for 48 hours) suggesting that elafin expression in this experiment is at physiologically relevant levels (Fig 2A). Treatment of Rb positive breast cancer cell lines with Ad-elafin resulted in a modest growth inhibition, whereas treatment of Rb negative cell lines resulted in precipitous decline in cell number (Figure 2B). All three of the Rb expressing cell lines demonstrated a significant reduction in BrdU incorporation, while the Rb deficient cell lines demonstrated no significant change in BrdU incorporation (Figure 2C). However, examination of TUNEL positivity in these cells demonstrated that while none of the Rb positive cells succumbed to apoptosis that all three Rb negative cells underwent apoptosis in response to elafin expression (Figure 2D). The percent TUNEL positivity ranged between 22-30% of all Rb negative cells treated with Ad-elafin, compared to 1-5% of Ad-Luciferase (p= 0.0127-0.003). These results suggest that elafin overexpression results in growth inhibition in Rb positive cells and apoptosis in Rb negative breast cancer cells.

Figure 2. Expression of elafin in Rb negative breast cancer cells results in apoptosis.

Figure 2

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MCF-7, ZR75.1, T47D, MDA-MB-157, MDA-MB-436, and MDA-MB-468 cells were treated with PBS (P), adenoviral luciferase (L), or adenoviral elafin (E). (A) Cells were harvested 48 hrs post-treatment and subjected to western blot analysis with indicated antibodies. (B) Growth was monitored by trypan blue exclusion test every 24 hours for 96 hours. * Elafin expression caused a statistically significant difference in cell number. (C) Proliferation was assessed 48 hours post-treatment by measuring BrdU incorporation (D) Apoptosis was assessed 72 hours post-treatment by TUNEL assay. All experiments were repeated in triplicate.

Inhibition of caspase activity leads to attenuation of elafin-mediated apoptosis

We next examined if caspase activity is modulated by elafin in the Rb negative cells lines by evaluating the ability of Ad-elafin treatment to trigger cleavage of caspase 3 and PARP, two indicators of caspase mediated apoptosis (Figure 3). The Rb deficient cell line MDA-MB-468 was treated with PBS, Ad-luciferase, and Ad-elafin for the indicated times. Western blot analysis revealed cleavage caspase 3 and PARP suggesting the activation of caspases following the treatment of cells with Ad-elafin (Figure 3A). We next investigated if inhibition of caspase activity could negate elafin-induced apoptosis. To this end, MDA-MB-468 and MDA-MB-157 cells were treated with Ad-elafin in the presence of the pan-caspase inhibitor zVad-fmk, DMSO, or PBS and cell viability was examined over time. Both of these Rb negative cells lines demonstrated a statistically significant reduction in cell viability following the introduction of Ad-elafin when compared to PBS or Ad-luciferase at 72, 96, and 120 hours. However, the addition of zVad-fmk was able to significantly inhibit Ad-elafin induced apoptosis when compared to DMSO or PBS treatment (Figure 3B). These results suggest that elafin induces caspase dependent, apoptosis in Rb deficient breast cancer cells.

Figure 3. Inhibition of caspase activity leads to attenuation of elafin-mediated apoptosis.

Figure 3

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(A) MDA-MB-468 were treated with PBS (P), adenoviral luciferase (L), or adenoviral elafin (E) for indicated times, lysates were subjected to western blot analysis with indicated antibodies. (B) Treated cells were simultaneously incubated with PBS, DMSO, or 50 μM of zVad-fmk; viability was measure by MTT assay every 24 hours for 120 hours. Eight replicates from two independent experiments were compiled and expressed as a percentage of the PBS control. Statistical significance was calculated by the t-test for no treatment vs. elafin-alone (1), luciferase alone vs. elafin alone (2), and elafin + DMSO vs. elafin + zVad-fmk are displayed as a table.

Knockdown of Rb in MDA-MB-231 cells increases sensitivity to elafin-induced apoptosis

Next, we asked if there is a cause and effect relationship between Rb and elafin by evaluating the ability of Rb downregulation to sensitize MDA-MB-231 cells to apoptosis when challenged with elafin. MDA-MB-231 cells expressing Rb shRNA (RBKD) cells were evaluated by western blot analysis, demonstrating efficient knockdown of Rb and expression of the elafin transgene (Fig 4A). Treatment of the RBKD cells with Ad-elafin resulted in a significant reduction in cell number 72 and 96 hours post-treatment when compared to MDA-MB-231 parental or shRNA clones (Fig 4A, 4B). TUNEL staining demonstrates a significant increase in the percentage of apoptotic cells in RBKD cells when compared to control cell lines (Figure 4C). These results illustrate a direct role for Rb deficiency in sensitizing breast cancer cells to elafin-induced apoptosis.

Figure 4. Knockdown of Rb in MDA-MB-231 cells increases sensitivity to elafin-induced apoptosis.

Figure 4

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MDA-MB-231 parental, shRNA control, and RBKD cell lines were treated with PBS (P), adenoviral luciferase (L), or adenoviral elafin (E). (A) Lysates were collected at 48 hours and subjected to western blot analysis. Growth was monitored by trypan blue exclusion test every 24 hours for 96 hours. Statistical significance was calculated by the t-test (B) Cell number at 96 hours was compared for all three cell lines as a percentage of the PBS control. (C) Apoptosis was assessed 72 hours post-treatment by TUNEL assay. All experiments were repeated in triplicate.

Rb is required for elafin induced G0/G1 arrest in MCF-7 cells

We have shown that treatment of Rb positive breast cancer cell lines with elafin causes them to undergo proliferative arrest not apoptosis (Figure 2). We next set out to decipher the role of elafin in mediating cell cycle arrest in Rb positive breast cancer cell lines by modulating the expression of both elafin and Rb and assessing the downstream consequences on cell proliferation and apoptosis (Figure 5& 6). When we treated MCF-7 (Rb positive) cells with either Ad-luciferase or Ad-elafin and subjected them to cell cycle analysis we found that overexpression of elafin causes cells to accumulate in the G1/G0 phase of the cell cycle and nearly eliminates the S-phase population by 72 hours (Fig 5A). We next examined if elafin overexpression modulates the phosphorylation of Rb and the kinase activity of CDK4 in the Rb positive MCF-7 breast cancer cells. For these experiments MCF-7 cells were treated with PBS, Ad-luciferse, or Ad-elafin for the indicated time periods and subjected to western blot and kinase assays. Elafin treatment resulted in a decrease in the phosphorylation of Rb using phospho-specific antibodies, directed against serine 807/811, serine 780, and total phosphorylated Rb (Figure 5B). The CDK4 kinase activity was measured using GST-Rb as a substrate, and showed a profound decrease in the kinase activity of CDK4 following overexpression of elafin, at 24, 48, and 72 hours (Figure 5C). These results suggest that the expression of elafin in MCF-7 cells causes a G0/G1 arrest characterized by a decreased in Rb phosphorylation, in part due to attenuation of CDK-4 kinase activity.

Figure 5. Expression of elafin in MCF-7 cells causes a G0/G1 arrest.

Figure 5

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(A) MCF-7 cells were infected with adenoviral luciferase or adenoviral elafin. DNA content was analyzed by propidium iodide staining, cell cycle distribution was calculated using the Dean-Jett-Fox model. (B-C) MCF-7 cells were treated with PBS (P), adenoviral luciferase (L), or adenoviral elafin (E). (B) Lysates were subjected to western blot analysis and probed with indicated antibodies. (C) CDK-4 was immunopercipitated from 250 μg of protein lysates, the immunocomplex was then subjected to in vitro kinase assay using GST-Rb as a substrate.

Figure 6. Rb is required for elafin induced G0/G1 arrest in MCF-7 cells.

Figure 6

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MCF-7 shRNA control and RBKD cell lines were treated with PBS (P), adenoviral luciferase (L), or adenoviral elafin (E). (A) Lysates were collected 48 hours post-treatment and analyzed by western blot. Growth was monitored by trypan blue exclusion test every 24 hours for 120 hours. Elafin expression caused a statistically significant difference in cell number at 96 hours only in the MCF-7 shRNA control cells (p<0.0001 vs. PBS and p=0.0001 vs Ad-luc). (B) Cell number at 96 hours were compared for MCF-7 parental (refer to Figure 4A), shRNA control, and RBKD cell lines as a percentage of the PBS control. (C) Proliferation was assessed 48 hours post-treatment by measuring BrdU incorporation. (D) MCF-7 p53KD and siRNA controls were treated with PBS (P), adenoviral luciferase (L), or adenoviral elafin (E). Lysates were collected at 48 hours and analyzed by western blot. Elafin overexpression caused a statistically significant difference in cell number at 96 hours in both the MCF-7 p53KD (p<0.0001 vs. PBS and p<0.0001 vs Ad-luc) and MCF-7 siRNA control cells (p<0.0001 vs. PBS and p<0.0001 vs Ad-luc). (E) Proliferation was assessed 48 hours post-treatment by measuring BrdU incorporation. All experiments were repeated in triplicate.

Next, we set out to examine if Rb downregulation in MCF-7 cells is sufficient to convert cell fate from growth arrest to apoptosis following elafin expression (as was the case in the Rb negative MDA-MB-231 cells-Fig 4). To accomplish this we treated MCF-7 cells stably expressing an shRNA vector targeting Rb with PBS, Ad-luciferase, and Ad-elafin and followed cell proliferation over 96 hours. Efficient Rb knockdown and consistent expression of the elafin transgene were confirmed by western blot analysis (Figure 6A). MCF-7 shRNA control cells treated with Ad-elafin demonstrated a moderate reduction in cell number at 96 hours when compared to controls (Figure 6A), similar to the pattern seen in the MCF-7 parental cell line (Figure 5A). However, MCF-7 RBKD cells showed no significant change in growth kinetics following treatment with Ad-elafin (Figure 6A). In fact, comparison of the parental, shRNA control and RBKD cell lines illustrates a significant reduction in the sensitivity of RBKD cells to elafin-induced cell cycle arrest (Figure 6B). This indicates that knockdown of Rb in MCF-7 cells does not sensitize cells to apoptosis, but is sufficient to attenuate cell cycle arrest. Consequently, MCF-7 RBKD cells expressing elafin have a significantly reduced BrdU incorporation as compared to shRNA controls and parental MCF-7 cells (Figure 6C).

Collectively, these results show that elafin induced growth arrest in breast cancer cells is Rb-dependent. We next explored a role for p53 in elafin mediated growth arrest. Using MCF-7 cells stably expressing a vector generating short oglionucleotides (siRNA) against p53 (Figure 6D), we show that overexpression of elafin caused a significant reduction in cell number at 96 hours, identical to the control cell line (Figure 6D), and the MCF-7 parental cell line (Figure 4A). BrdU incorporation following Ad-elafin treatment showed no statistically significant difference in the elafin-mediated inhibition of BrdU incorporation when compared to control cell lines (Figure 6E). These results indicate that elafin overexpression causes Rb dependent cell cycle arrest in MCF-7 cells, independently of p53 activity.

Overexpression of elafin causes apoptosis in MCF-7 cells only after knockdown of Rb and restoration of caspase 3

Downregulation of Rb was shown to sensitize MDA-MB-231 cells to elafin induced apoptosis (Figure 4), however downregulation of Rb in MCF-7 cells was unable to replicate these results (Figure 6). MCF-7 cells lack endogenously expressed caspase 3, which is required for elafin induced apoptosis (Figure 3). This led to the hypothesis that loss of Rb and presence of caspase 3 are both required for elafin-induced apoptosis in MCF-7 cells. To address this hypothesis, we first confirmed that elafin expression fails to induce apoptosis following knockdown of Rb alone or reconstitution of caspase 3 alone. MCF-7 RBKD cells, described in figure 6, and MCF-7 caspase 3 reconstituted cells were treated with PBS, Adluciferase, and Ad-elafin and apoptosis was measured by TUNEL assay revealing no significant increase in apoptotic cell death under either condition (Fig 7A).

Figure 7. Overexpression of elafin causes apoptosis in MCF-7 cells only after knockdown of Rb and restoration of caspase 3.

Figure 7

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(A) MCF-7 RBKD, shRNA control cells, MCF-7 pcDNA3.1 empty vector and pcDNA3.1-caspase 3 expressing cells were treated with PBS (P), adenoviral luciferase (L), or adenoviral elafin (E). Lysates from MCF-7 pcDNA3.1 empty vector and pcDNA3.1-caspase 3 expressing cells subjected to western blot analysis 48 hours post treatment (right panels). Apoptosis was assessed by TUNEL assay (left panels). (B) Stable clones and a stable pool were generated on the MCF-7 RBKD background expressing either pcDNA3.1 empty vector or pcDNA3.1-caspase 3. Cells were treated with PBS (P), adenoviral luciferase (L), or adenoviral elafin (E). Lysates were collected from each cell line and subjected to western blot analysis with the indicated antibodies. PBS treated cells were used to assess the levels of Rb and caspase 3 and MDA-MB-231 lysate was used as a control for caspase 3 expression. (C) Viability was measured by MTT assay every 24 hours for 120 hours and calculated at each time point by normalizing values from luciferase and elafin treated cells to PBS control then plotting the difference between the viability of elafin and luciferase (i.e. elafin effect – viral effect). (D-E) Stable clones and a stable pool were also generated on the MCF-7 control shRNA background expressing either pcDNA3.1 empty vector or pcDNA3.1-caspase 3. These cells were assayed in the same manner as panels B and C. (F) The viabilities measured at 120 hours in the cell lines generated from the MCF-7 RBKD and MCF-7 control shRNA as well as the parental cell lines were pooled and statistically compared. (F) Apoptosis was measured by TUNEL assay.

To directly examine if Rb downregulation and presence of caspase 3 are required for apoptosis in MCF-7 cells following elafin expression we established clones of both the MCF-7 RBKD and MCF-7 shRNA control cell lines expressing either the pcDNA3.1 backbone vector or pcDNA3.1-caspase 3 vector. Knockdown of Rb, stable expression of caspase 3, and expression of the elafin transgene were verified by western blot analysis (Figure 7B). Treatment of MCF-7 RBKD cells with Ad-elafin caused a severe reduction in cell viability following reconstituted caspase 3 (Figure 7C). The same experiment was preformed in MCF-7 shRNA control cells line, however in these cells reconstitution of caspase 3 did not lead to a significant decrease in viability compared to controls (Figure 7D and E). Comparison of cell viability at 120 hours post treatment demonstrates that elafin mediated loss of cell viability in MCF-7 RBKD clones expressing caspase 3 is significantly different from all other MCF-7 cell lines examined (Figure 7F-compare RBKD CASP3 to the other cell population). To determine if this reduced viability occurs as a result of increased apoptosis, TUNEL assay was performed on elafin expressing MCF-7 RBKD cells stably expressing either caspase 3 or the pcDNA3.1 backbone (Fig 7G). These results revealed a significant increase in the percentage of cells undergoing apoptosis in MCF-7 RBKD expressing caspase 3 versus controls (PCDNA + Ad-luc, PCDNA+Ad-Elafin or CASP 3 + Ad-Luc). These results indicate that loss of Rb and the reconstitution of caspase 3 are both required for elafin-induced apoptosis in the MCF-7 cell line. T47D cells are Rb positive and endogenously express caspase 3, therefore we tested the validity of our observation in these cells by knockdown of Rb using siRNA and subsequent treatment with PBS, Ad-luciferase, and Adelafin. These analyses reveal that knockdown of Rb is sufficient to sensitize a caspase 3 positive cell line to elafin-mediated apoptosis (Supplementary Figure 1).

Discussion

Elafin is expressed in HMECs, but it is transcriptionally downregulated in tumor derived cell lines (9). In this study, we show that expression of elafin causes cell cycle arrest or apoptosis in breast tumor derived cell lines depending on Rb status, but does not affect normal HMECs with an intact G1/S checkpoint. This suggests that elafin has novel anti-tumor properties in breast cancer and represents a candidate therapeutic capable of specifically targeting tumor cells with disruption of the G1/S checkpoint with no toxicity in normally dividing cells.

We initially observed that expression of elafin in HMECs lacking Rb (76NE7) caused these cells to undergo apoptotic cell death. Such a Rb-dependent apoptotic affect led us to hypothesize that tumor cells with a deregulated Rb pathway, could also be forced to undergo apoptosis following elafin expression. Indeed, we observed that the expression of elafin can induce apoptosis in Rb negative cells, but requires caspase 3 activation. Breast cancer cells, which are Rb-negative, but do not express caspase 3, are resistant to apoptosis. We also found that Rb-positive breast cancer cells are growth inhibited in response to elafin expression.

The loss of elafin in breast cancer cell lines suggests a tumor suppressive role for elafin in the mammary gland. There is precedent for the existence of a tumor suppressor serine protease inhibitor that is downregulated in breast cancer cells. Maspin, a member of the serpin family of serine protease inhibitors, is differentially expressed in normal mammary epithelial cells and breast tumor cells by epigenetic processes (20). Since its identification, maspin has emerged as a tumor suppressor in breast, prostate, and ovarian cancers with diverse roles in angiogenesis, tumor invasion, apoptosis and metastasis (21-23). Dissimilar from elafin, a protease target has not been identified for maspin. Elafin is a potent inhibitor of the serine protease, human neutrophil elastase (HNE), which has an established role during tumorigenesis in the breast and other tissue types (24-27). HNE is an independent prognostic marker in breast cancer and has been correlated with poor prognosis, increased metastasis, and resistance to chemotherapy (24, 25, 28-31). In experimental models, pharmacological HNE inhibitors were able to attenuate the development of skin tumors, reduce growth and metastasis in a lung xenograft model, and inhibit proliferation/chemotaxis of pancreatic cells (32-35). Several HNE substrates with relevance to tumorigenesis have been described, including insulin receptor substrate 1 (IRS-1), cyclin E and cut-like homeobox 1(CUX-1) (36-40). Given the intracellular functions of HNE in promoting tumorigenesis, elafin may be a critical component of intracellular control of HNE in mammary epithelial cells. Our experimental re-introduction of elafin into breast tumor cells suggests that the pathways deregulated by HNE may be critical to proliferation and survival. In particular, elafin toxicity in tumor cells lacking Rb suggests that HNE is required to modulate the apoptotic effect of E2F transcription factors.

Supplementary Material

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Acknowledgements

We thank Bingliang Fang, Xinbin Chen, and Eric Knudson, for reagents; Wendy Schober for flow cytometry analysis; Said Akli, for critical discussions during the course of this study. This work was supported by National Institutes of Health grant CA87458 and National Cancer Institute grant P50CA116199 and a grant from Clayton Foundation to Khandan Keyomarsi and by NCI # CA16672 MDACC CCSG.

References

  • 1.Wiedow O, Schroder JM, Gregory H, Young JA, Christophers E. Elafin: an elastase-specific inhibitor of human skin. Purification, characterization, and complete amino acid sequence. J Biol Chem. 1990;265:14791–5. [PubMed] [Google Scholar]
  • 2.Balloy V, Sallenave JM, Crestani B, Dehoux M, Chignard M. Neutrophil DNA contributes to the antielastase barrier during acute lung inflammation. Am J Respir Cell Mol Biol. 2003;28:746–53. doi: 10.1165/rcmb.2002-0119OC. [DOI] [PubMed] [Google Scholar]
  • 3.Schmid M, Fellermann K, Fritz P, Wiedow O, Stange EF, Wehkamp J. Attenuated induction of epithelial and leukocyte serine antiproteases elafin and secretory leukocyte protease inhibitor in Crohn's disease. J Leukoc Biol. 2007;81:907–15. doi: 10.1189/jlb.0906581. [DOI] [PubMed] [Google Scholar]
  • 4.Yamamoto S, Egami H, Kurizaki T, et al. Immunohistochemical expression of SKALP/elafin in squamous cell carcinoma of the oesophagus. Br J Cancer. 1997;76:1081–6. doi: 10.1038/bjc.1997.511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Alkemade HA, Molhuizen HO, van Vlijmen-Willems IM, van Haelst UJ, Schalkwijk J. Differential expression of SKALP/Elafin in human epidermal tumors. Am J Pathol. 1993;143:1679–87. [PMC free article] [PubMed] [Google Scholar]
  • 6.Alkemade HA, van Vlijmen-Willems IM, van Haelst UJ, van de Kerkhof PC, Schalkwijk J. Demonstration of skin-derived antileukoproteinase (SKALP) and its target enzyme human leukocyte elastase in squamous cell carcinoma. J Pathol. 1994;174:121–9. doi: 10.1002/path.1711740208. [DOI] [PubMed] [Google Scholar]
  • 7.Westin U, Nystrom M, Ljungcrantz I, Eriksson B, Ohlsson K. The presence of elafin, SLPI, IL1-RA and STNFalpha RI in head and neck squamous cell carcinomas and their relation to the degree of tumour differentiation. Mediators Inflamm. 2002;11:7–12. doi: 10.1080/09629350210304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Zhang M, Zou Z, Maass N, Sager R. Differential expression of elafin in human normal mammary epithelial cells and carcinomas is regulated at the transcriptional level. Cancer Res. 1995;55:2537–41. [PubMed] [Google Scholar]
  • 9.Yokota T, Bui T, Liu Y, Yi M, Hunt KK, Keyomarsi K. Differential regulation of elafin in normal and tumor-derived mammary epithelial cells is mediated by CCAAT/enhancer binding protein beta. Cancer Res. 2007;67:11272–83. doi: 10.1158/0008-5472.CAN-07-2322. [DOI] [PubMed] [Google Scholar]
  • 10.Knudsen ES, Knudsen KE. Tailoring to RB: tumour suppressor status and therapeutic response. Nat Rev Cancer. 2008;8:714–24. doi: 10.1038/nrc2401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Pietilainen T, Lipponen P, Aaltomaa S, Eskelinen M, Kosma VM, Syrjanen K. Expression of retinoblastoma gene protein (Rb) in breast cancer as related to established prognostic factors and survival. Eur J Cancer. 1995;31A:329–33. doi: 10.1016/0959-8049(94)00463-f. [DOI] [PubMed] [Google Scholar]
  • 12.Herschkowitz JI, He X, Fan C, Perou CM. The functional loss of the retinoblastoma tumour suppressor is a common event in basal-like and luminal B breast carcinomas. Breast Cancer Res. 2008;10:R75. doi: 10.1186/bcr2142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Trere D, Brighenti E, Donati G, et al. High prevalence of retinoblastoma protein loss in triple-negative breast cancers and its association with a good prognosis in patients treated with adjuvant chemotherapy. Ann Oncol. 2009 doi: 10.1093/annonc/mdp209. [DOI] [PubMed] [Google Scholar]
  • 14.Sorlie T, Perou CM, Tibshirani R, et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A. 2001;98:10869–74. doi: 10.1073/pnas.191367098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Carey LA, Dees EC, Sawyer L, et al. The triple negative paradox: primary tumor chemosensitivity of breast cancer subtypes. Clin Cancer Res. 2007;13:2329–34. doi: 10.1158/1078-0432.CCR-06-1109. [DOI] [PubMed] [Google Scholar]
  • 16.Band V, Sager R. Distinctive traits of normal and tumor-derived human mammary epithelial cells expressed in a medium that supports long-term growth of both cell types. Proc Natl Acad Sci U S A. 1989;86:1249–53. doi: 10.1073/pnas.86.4.1249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Band V, Zajchowski D, Kulesa V, Sager R. Human papilloma virus DNAs immortalize normal human mammary epithelial cells and reduce their growth factor requirements. Proc Natl Acad Sci U S A. 1990;87:463–7. doi: 10.1073/pnas.87.1.463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Rao S, Lowe M, Herliczek TW, Keyomarsi K. Lovastatin mediated G1 arrest in normal and tumor breast cells is through inhibition of CDK2 activity and redistribution of p21 and p27, independent of p53. Oncogene. 1998;17:2393–402. doi: 10.1038/sj.onc.1202322. [DOI] [PubMed] [Google Scholar]
  • 19.McGahren-Murray M, Terry NH, Keyomarsi K. The differential staurosporine-mediated G1 arrest in normal versus tumor cells is dependent on the retinoblastoma protein. Cancer Res. 2006;66:9744–53. doi: 10.1158/0008-5472.CAN-06-1809. [DOI] [PubMed] [Google Scholar]
  • 20.Domann FE, Rice JC, Hendrix MJ, Futscher BW. Epigenetic silencing of maspin gene expression in human breast cancers. Int J Cancer. 2000;85:805–10. doi: 10.1002/(sici)1097-0215(20000315)85:6<805::aid-ijc12>3.0.co;2-5. [DOI] [PubMed] [Google Scholar]
  • 21.Zhang M, Volpert O, Shi YH, Bouck N. Maspin is an angiogenesis inhibitor. Nat Med. 2000;6:196–9. doi: 10.1038/72303. [DOI] [PubMed] [Google Scholar]
  • 22.Zou Z, Anisowicz A, Hendrix MJ, et al. Maspin, a serpin with tumor-suppressing activity in human mammary epithelial cells. Science. 1994;263:526–9. doi: 10.1126/science.8290962. [DOI] [PubMed] [Google Scholar]
  • 23.Jiang N, Meng Y, Zhang S, Mensah-Osman E, Sheng S. Maspin sensitizes breast carcinoma cells to induced apoptosis. Oncogene. 2002;21:4089–98. doi: 10.1038/sj.onc.1205507. [DOI] [PubMed] [Google Scholar]
  • 24.Akizuki M, Fukutomi T, Takasugi M, et al. Prognostic significance of immunoreactive neutrophil elastase in human breast cancer: long-term follow-up results in 313 patients. Neoplasia. 2007;9:260–4. doi: 10.1593/neo.06808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Foekens JA, Ries C, Look MP, Gippner-Steppert C, Klijn JG, Jochum M. The prognostic value of polymorphonuclear leukocyte elastase in patients with primary breast cancer. Cancer Res. 2003;63:337–41. [PubMed] [Google Scholar]
  • 26.Lane AA, Ley TJ. Neutrophil elastase cleaves PML-RARalpha and is important for the development of acute promyelocytic leukemia in mice. Cell. 2003;115:305–18. doi: 10.1016/s0092-8674(03)00852-3. [DOI] [PubMed] [Google Scholar]
  • 27.Porter DC, Zhang N, Danes C, et al. Tumor-specific proteolytic processing of cyclin E generates hyperactive lower-molecular-weight forms. Mol Cell Biol. 2001;21:6254–69. doi: 10.1128/MCB.21.18.6254-6269.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Foekens JA, Ries C, Look MP, Gippner-Steppert C, Klijn JG, Jochum M. Elevated expression of polymorphonuclear leukocyte elastase in breast cancer tissue is associated with tamoxifen failure in patients with advanced disease. Br J Cancer. 2003;88:1084–90. doi: 10.1038/sj.bjc.6600813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Yamashita J, Ogawa M, Shirakusa T. Free-form neutrophil elastase is an independent marker predicting recurrence in primary breast cancer. J Leukoc Biol. 1995;57:375–8. doi: 10.1002/jlb.57.3.375. [DOI] [PubMed] [Google Scholar]
  • 30.Foekens J, Ries C, Look M, Gippner-Steppert C, Klijn J, Jochum M. The prognostic value of polymorphonuclear leukocyte elastase in patients with primary breast cancer. [PubMed]
  • 31.Sato T, Takahashi S, Mizumoto T, et al. Neutrophil elastase and cancer. Surg Oncol. 2006;15:217–22. doi: 10.1016/j.suronc.2007.01.003. [DOI] [PubMed] [Google Scholar]
  • 32.Starcher B, O'Neal P, Granstein RD, Beissert S. Inhibition of neutrophil elastase suppresses the development of skin tumors in hairless mice. J Invest Dermatol. 1996;107:159–63. doi: 10.1111/1523-1747.ep12329559. [DOI] [PubMed] [Google Scholar]
  • 33.Kamohara H, Sakamoto K, Mita S, An XY, Ogawa M. Neutrophil elastase inhibitor (ONO-5046.Na) suppresses the proliferation, motility and chemotaxis of a pancreatic carcinoma cell line, Capan-1. Res Commun Mol Pathol Pharmacol. 1997;98:103–8. [PubMed] [Google Scholar]
  • 34.Inada M, Yamashita J, Ogawa M. Neutrophil elastase inhibitor (ONO-5046-Na) inhibits the growth of human lung cancer cell lines transplanted into severe combined immunodeficiency (scid) mice. Res Commun Mol Pathol Pharmacol. 1997;97:229–32. [PubMed] [Google Scholar]
  • 35.Inada M, Yamashita J, Nakano S, Ogawa M. Complete inhibition of spontaneous pulmonary metastasis of human lung carcinoma cell line EBC-1 by a neutrophil elastase inhibitor (ONO-5046.Na). Anticancer Res. 1998;18:885–90. [PubMed] [Google Scholar]
  • 36.Keyomarsi K, Tucker SL, Buchholz TA, et al. Cyclin E and survival in patients with breast cancer. N Engl J Med. 2002;347:1566–75. doi: 10.1056/NEJMoa021153. [DOI] [PubMed] [Google Scholar]
  • 37.Akli S, Van Pelt CS, Bui T, et al. Overexpression of the low molecular weight cyclin E in transgenic mice induces metastatic mammary carcinomas through the disruption of the ARF-p53 pathway. Cancer Res. 2007;67:7212–22. doi: 10.1158/0008-5472.CAN-07-0599. [DOI] [PubMed] [Google Scholar]
  • 38.Akli S, Zheng PJ, Multani AS, et al. Tumor-Specific Low Molecular Weight Forms of Cyclin E Induce Genomic Instability and Resistance to p21, p27, and Antiestrogens in Breast Cancer. Cancer Res. 2004;64:3198–208. doi: 10.1158/0008-5472.can-03-3672. [DOI] [PubMed] [Google Scholar]
  • 39.Goulet B, Markovic Y, Leduy L, Nepveu A. Proteolytic processing of cut homeobox 1 by neutrophil elastase in the MV4;11 myeloid leukemia cell line. Mol Cancer Res. 2008;6:644–53. doi: 10.1158/1541-7786.MCR-07-0268. [DOI] [PubMed] [Google Scholar]
  • 40.Houghton AM, Rzymkiewicz DM, Ji H, et al. Neutrophil elastase-mediated degradation of IRS-1 accelerates lung tumor growth. Nat Med. doi: 10.1038/nm.2084. [DOI] [PMC free article] [PubMed] [Google Scholar]

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