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[Cancer Research 65, 8853-8860, October 1, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Stable RNA Interference–Mediated Suppression of Cyclophilin A Diminishes Non–Small-Cell Lung Tumor Growth In vivo

Brandon A. Howard1,2, Ryohei Furumai3, Michael J. Campa1, Zahid N. Rabbani4, Zeljko Vujaskovic4, Xiao-Fan Wang3 and Edward F. Patz, Jr.1,2,3

Departments of 1 Radiology, 2 Pathology, 3 Pharmacology and Cancer Biology, and 4 Radiation Oncology, Duke University Medical Center, Durham, North Carolina

Requests for reprints: Edward F. Patz, Jr., Department of Radiology, Duke University Medical Center, Box 3808, Durham, NC 27710. Phone: 919-684-7311; Fax: 919-684-7165; E-mail: patz0002{at}mc.duke.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cyclophilin A (CypA) was recently reported to be overexpressed in non–small-cell lung cancer, and represents a potentially novel therapeutic target. To determine the role of CypA in oncogenesis, stable RNA interference (RNAi)–mediated knockdown of CypA was established in two non–small-cell lung cancer cell lines (ADLC-5M2 and LC-103H), and these cells were grown as xenografts in severe combined immunodeficient mice. Tumor cell proliferation, apoptosis, and angiogenesis were measured by Ki67, terminal deoxyribonucleotidyl transferase–mediated dUTP nick-end labeling, and CD31 immunohistochemistry, respectively. Tumor glucose metabolism was assessed by fluorodeoxyglucose positron emission tomography imaging. Knockdown of CypA correlated in vivo with slower growth, less fluorodeoxyglucose uptake, decreased proliferation, and a greater degree of apoptosis in the tumors. These results establish the relevance of CypA to tumor growth in vivo, specifically to proliferation and apoptosis. Elucidation of the precise role of CypA in these pathways may lead to new targeted therapies for lung cancer.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lung cancer is the leading cause of cancer death, and more than 170,000 new cases are diagnosed each year in the United States alone. Despite extensive research efforts in lung cancer screening, diagnostics, and therapeutics, the overall 5-year survival remains at ~14%. Therefore, in an effort to identify novel molecular targets for focused therapy, we previously compared protein expression profiles between non–small-cell lung cancer and normal lung, and identified overexpressed tumor proteins (1). One of these proteins, cyclophilin A (CypA), was found to have >7-fold overexpression in the tumor, although its role in lung cancer is unknown.

CypA is found in normal cells and comprises ~0.1% of cytoplasmic protein (2). It is a member of the peptidyl-prolyl isomerases, a group of proteins that catalyzes cis-trans isomerization about peptidyl-prolyl bonds during protein folding or conformational changes. CypA was first identified as the primary intracellular target of the immunosuppressant cyclosporin A (CsA; ref. 3). Through inhibition of the phosphatase calcineurin, CypA:CsA down-regulates the nuclear translocation of nuclear factors of activated T cells and downstream gene transcription (i.e., interleukin 2 in T cells; ref. 4).

Recent evidence, however, points to possible roles for CypA overexpression in tumorigenesis. In two separate reports, CypA was shown to interact with the retinoblastoma susceptibility gene product p105Rb (Rb) in Jurkat T-cell extracts. Nuclear translocation of Rb:CypA correlated with retinoic acid–induced neural differentiation (5, 6). Although CypA seems to have a role in apoptosis, it is not clear if it is involved in activation or inhibition, or both. CypA was shown to be involved in caspase activation in neurons (7), and to cooperate with apoptosis-inducing factor during apoptosis-associated chromatinolysis (8). However, CypA was also reported to bind the calcium-sequestering protein calreticulin by yeast two-hybrid screen (9), which suggests that it may inhibit calcium fluxes required for apoptosis (10).

We recently examined the expression of CypA in 234 primary non–small-cell lung cancer specimens by tissue microarray immunohistochemistry, and although we did not find a correlation with survival, we found that CypA was strongly expressed (intensity grade 2 or greater) by a majority of tumors. This ubiquitous overexpression in lung cancer suggests that CypA could be an important component of neoplastic transformation (11).

The purpose of the present study was to determine the effect of RNA interference (RNAi)–mediated knockdown of CypA expression on tumor growth in vivo, and to gain insight into the relevance of CypA in lung cancer biology.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
The lung adenocarcinoma ADLC-5M2 (hereafter referred to as 5M2) and large cell carcinoma LC-103H cell lines were previously characterized and shown to be tumorigenic in athymic nude mice (12). These lines were maintained in RPMI 1640/10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA). Population doubling times were measured by counting the cells in a hemacytometer and the number of viable cells was determined by Trypan blue exclusion. Both cell lines were known to express CypA by Western blot (data not shown). The transformed small airway epithelial line S1LEK3 was a gift from Robert Weinberg (Department of Biology, Massachusetts Institute of Technology, Cambridge, MA) and was maintained in DMEM/10% FBS as previously described (13).

Vector Construction
RNA interference vector construction. The pSUPER.retro plasmid containing puromycin and ampicillin resistance genes was purchased from Oligoengine (Seattle, WA) and linearized with HindIII. Oligonucleotides designed to produce a hairpin siRNA containing either (a) a cyclophilin siRNA sequence (Ambion, Austin, TX) with no homology to other human genes besides CypA, as determined by nucleotide-nucleotide BLAST search, or (b) a scrambled sequence with no homology to other genes were annealed and ligated into the linearized plasmid using T4 DNA ligase (Invitrogen). Chemically competent DH5{alpha} E. coli were transformed and positive transformants were isolated by ampicillin selection (100 µg/mL) and amplified using standard methods. Presence of insert-containing pSUPER.retro was confirmed by EcoRI/HindIII double digest of the plasmid DNA isolated from several bacterial colonies. Plasmid DNA from positive clones was isolated using a QIAGEN (Valencia, CA) Midi-prep kit and sequenced for additional verification.

Cyclophilin A overexpression vector construction. Human CypA cDNA (Stratagene, La Jolla, CA) was cloned into the pFLAG-CMV2 expression vector (Kodak, Rochester, NY) by standard methods and verified by sequencing.

Transfections
5M2 cells were grown to 60% confluency in six-well plates and transfected with 2 µg pSUPER.retro-CypA siRNA or scrambled siRNA using Lipofectamine (Invitrogen) per instructions of the manufacturer. At 48 hours following transfection, cells were passaged into medium containing 2 µg/mL puromycin and allowed to grow until distinct colonies could be distinguished. Ten single colonies of CypA- or scrambled-vector 5M2 transfectants (named C1, C2, etc., and S1, S2, etc., respectively) were isolated and expanded. For transfection of the LC-103H cells, 293T cells were transfected with pSUPER.retro-CypA siRNA and the pCL retroviral packaging vector to produce retroviral supernatants. LC-103H cells were infected with the retroviral supernatants and selected with 2 µg/mL puromycin for 2 weeks. Following selection, the polyclonal population (LC-103H-KD) was used in subsequent experiments. For overexpression experiments, S1LEK3 cells were cotransfected with 1 µL of pBabe-bleo with 18 µL FuGENE (Roche, Indianapolis, IN) and 5 µL of either pFLAG-CMV2-CypA or pFLAG-CMV2 vector alone. Cells were selected by 100 µg/mL Zeocin (Invitrogen) for 4 weeks. For subsequent experiments, single clones were isolated and expanded from the S1LEK3-CypA–transfected cells (S1LEK-OE), and a polyclonal population was obtained from the S1LEK3-vector–transfected cells (S1LEK3-Ve).

Xenograft Formation
All animal work described herein was approved by the Duke Institutional Animal Care and Use Committee. Following determination of absence of mycoplasma contamination by nucleic acid hybridization, 3 million cells each of three 5M2-CypA knockdown single clones (C1, C2, and C4), 5M2-scrambled vector (S5), and parental 5M2 were injected in 200 µL RPMI 1640/10% FBS s.c. into the flank of 4-week-old, male, severe combined immunodeficient/beige mice (Charles River Laboratories, Wilmington, MA); four animals per cell line were used. For the S1LEK3 cells, 2 million cells were injected. For the LC-103H-KD and LC-103H parental cells, 3 million cells were injected. Tumor dimensions were measured every 2 to 3 days with calipers and the volume was calculated by the formula V = 0.5 x L x W2, where L is long diameter and W is short diameter. Mice harboring the C1, C2, C4, S5, and 5M2 xenografts were sacrificed at 30 days and tumors and organs (liver, lungs, rib, spleen, and blood) were harvested for subsequent analysis by fixing half of each specimen in buffered neutral formalin and snap-freezing the remaining half in liquid nitrogen. Mice with the LC-103H-KD and LC-103H xenografts were sacrificed at 34 days and tumors were harvested as described above.

Preparation of Tissue Extracts
Tissue specimens were minced and incubated in 10 mL PBS at 4°C for 30 minutes with gentle agitation to minimize blood contamination. Five volumes of Mammalian Protein Extraction Reagent (Pierce, Rockford, IL) containing 1x Complete Mini Protease Inhibitor Cocktail (Roche) were added to the tissue and the mixture was rotated for 30 minutes at 4°C. After a freeze-thaw cycle, the tissue was homogenized with a pestle and rotated for 1 hour at 4°C. For cell line lysis, each cell pellet isolated from a confluent 10 cm dish was resuspended in 0.5 mL Mammalian Protein Extraction Reagent/Protease Inhibitor and incubated for 30 minutes at 4°C. Tissue homogenates or cell lysates were centrifuged at 16,000 x g for 30 minutes at 4°C to remove cellular debris. Protein content of the supernatant fraction was then quantitated by a Bradford dye binding assay (Bio-Rad, Hercules, CA) using bovine serum albumin as standard.

Western Blot
Lysates were subjected to one-dimensional SDS-PAGE on a 4% to 20% polyacrylamide gel, and proteins were electroblotted to polyvinylidene difluoride membrane. Membranes were probed with anti-human CypA polyclonal rabbit serum (CellTech Systems, Norcross, GA) at 1:50,000 dilution and mouse pan-actin monoclonal antibody (NeoMarkers, Fremont, CA) at 0.2 µg/mL in 5% (w/v) nonfat dry milk in TBS (50 mmol/L Tris-HCl, 138 mmol/L NaCl, pH 7.6). Bound antibody was detected with horseradish peroxidase (HRP)–conjugated anti-rabbit immunoglobulin G (IgG) and anti-mouse IgG secondary antibodies using SuperSignal West Femto chemiluminescent detection system (Pierce).

Immunohistochemistry
Immunohistochemistry was done using 8 to 10 µm serial sections of frozen tissue placed onto positively charged glass slides using a single-staining procedure. The protocols used with each antibody are given below.

Microvessel density (CD31). Microvessel counting was used to assess angiogenesis. Frozen sections were fixed in 4°C acetone for 10 minutes. After endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 15 minutes, the slides were blocked with 10% donkey serum for 15 minutes. The slides were then incubated with the primary antibody, rat anti-mouse CD31 (BD Biosciences, San Jose, CA; dilution 1:50) for 1 hour at 41°C, and washed with PBS. Omission of the primary antibody served as negative control. Biotinylated donkey anti-rat antibody was applied for 30 minutes at room temperature, followed by application of an avidin:biotinylated HRP complex (Vectastain ABC kit, Vector Labs, Inc., Burlingame, CA). Color was developed by 5-minute incubation in NovaRed solution (Vector Labs). Slides were counterstained with hematoxylin. Microvessel density was calculated at lower magnification (x200) as follows: for each xenograft tumor, vessels were counted from four to five randomly chosen fields and averaged. The average of the averages within each cell line group was then computed.

Tumor cell proliferation was assessed by Ki67 immunoreactivity. Anti-Ki67 rabbit polyclonal antibody (Vector Labs) was applied to the slides at a dilution of 1:2,000 and incubated overnight at 4°C. The slides were then stained by the avidin-biotin method, as described above. 3,3'-Diaminobenzidine chromogen (Vector Labs) was used to develop brown color for 5 minutes. Slides were lightly counterstained with hematoxylin. Tumor cells were considered positive for the Ki67 antigen if there was intranuclear staining. After the slides were scanned at low magnification (40x objective), the cells with positively stained nuclei were counted in four to five random fields at a magnification of x400.

Terminal deoxyribonucleotidyl transferase–mediated dUTP nick-end labeling assay. In situ Cell Death Detection Kit, POD (Roche) was used to detect apoptosis and the instructions as described by manufacturer were followed with brief modifications. The slides were scanned at low magnification (x100), and the cells with positively stained nuclei were counted in four to five fields at a magnification of x400.

Serum Withdrawal
Knockdown (C1, C2, and C4) or control (S5 and parental 5M2) cells were plated in replicates of four at a density of 20,000 cells per well in 12-well plates in RPMI 1640/10% FBS and allowed to adhere overnight. The culture medium was then aspirated and replaced with RPMI 1640. Media was changed at day 6 to control for the effects of acidosis, micronutrient depletion, etc. Cells were counted on days 3, 6, and 9 and viable cells were identified by Trypan blue exclusion.

Thymidine Incorporation Assay
Fifty-thousand cells in 1 mL RPMI 1640/10% FBS were seeded in a 35 mm dish and allowed to incubate for 24 hours. [3H]Thymidine (Perkin-Elmer Life Analysis Sciences, Boston, MA) was then added in RPMI 1640/10% FBS to a final concentration of 5 µCi/mL, and the incubation was continued for an additional 4 hours. Cells were washed twice with PBS, then washed with 10% trichloroacetic acid, and allowed to incubate in 10% trichloroacetic acid for 1 hour at 4°C. Following a 15-minute incubation in 0.2% NaOH at 4°C, radioactivity was quantitated in a scintillation counter.

In vivo Fluorodeoxyglucose-Positron Emission Tomography Imaging
At 2 days before the tumor growth study end point, one mouse each from the C1 (CypA knockdown) and 5M2 (parental cell line) groups was anesthetized i.p. with ketamine and diazepam (100 and 5 mg/kg, respectively) and placed in the MicroPET scanner (Rodent 4, Concorde Microsystems, Knoxville, TN) in the Duke Center for In vivo Microscopy. [18F]FDG, 600 to 700 µCi, was injected via peritoneal catheter and emission scan data were acquired for 45 minutes. Following external calibration, the standardized uptake value (SUV) was calculated to allow a comparison of the metabolic activities in the tumors, by the formula SUV = mean activity in region of interest / (injected dose / mass of mouse).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cyclophilin A knockdown correlated with slower tumor growth in vivo. Stable transfection of 5M2 cells with pSUPER-CypA RNAi vector yielded ~90% CypA knockdown by Western blot as quantitated by chemiluminescence. One transfectant single clone, which had the greatest CypA knockdown, and two single clones, which had equal but lesser degrees of CypA knockdown (C4, C1, and C2, respectively), were used for subsequent experiments. Single clones transfected with pSUPER-scrambled RNAi showed no difference in CypA levels relative to parental 5M2 cells (Fig. 1A). This scrambled control was used to control for nonspecific effects of the RNAi vector. A similar degree of knockdown was observed with LC-103H cells transfected with pSUPER-CypA RNAi vector (data not shown).



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  		Figure 1. A, CypA knockdown of ~90% was achieved following transfection of 5M2 lung adenocarcinoma cells with pSUPER.retro-CypA RNAi. Western blot of 20 µg cell line lysate showing CypA knockdown. Transfection of pSUPER.retro-scrambled RNAi had no effect on CypA levels relative to parental 5M2. C1, C2, and C4, CypA knockdown clones; S5, scrambled clone. B, verification of persistence of CypA knockdown in vivo by Western blot. For each lane, 20 µg of xenograft tumor protein extract were subjected to anti-CypA Western blot.

 
When grown as xenografts in severe combined immunodeficient mice, CypA knockdown persisted (Fig. 1B) and CypA knockdown cells yielded slower-growing tumors than control cells for both the 5M2 and LC-103H lung cancer lines (Fig. 2A and B). At 30 days, tumors from CypA knockdown 5M2 cells were on average 72% smaller in volume than tumors from control cells (554 versus 1,944 mm3, respectively). At 34 days, tumors from the CypA knockdown LC-103H cells were on average 87% smaller than control tumors (997 versus 128 mm3, respectively). These results indicated that CypA knockdown had a significant effect on tumor growth in vivo.



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  		Figure 2. CypA knockdown correlates with slower tumor growth in vivo. A, mean tumor growth curves for CypA knockdown (C1, C2, and C4), scrambled (S5), and parental 5M2 xenografts. B, mean tumor growth curves for CypA knockdown (LC-103H KD) and parental (LC-103H) xenografts.

 
Cyclophilin A overexpression conferred a growth advantage in vivo. The effect of CypA overexpression was also examined. The small airway epithelial cell line S1LEK3, transformed with hTERT, SV40 large T antigen, and oncogenic ras, was transfected with a pFlag-CMV2-CypA overexpression construct. These S1LEK3 cells expressing exogenous CypA resulted in faster-growing xenografts in immunodeficient severe combined immunodeficient mice as compared with cells transfected with the empty vector (Fig. 3). At 21 days, large tumors were observed in the CypA-overexpressing group, whereas none was observed in the vector group. Moreover, tumors in the exogenous CypA group began growing at 10 days, whereas no tumors formed in the vector group until ~31 days.



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  		Figure 3. CypA overexpression correlates with faster tumor growth in vivo. Mean tumor growth curves for the transformed small airway epithelial line S1LEK3 transfected with either pCMV2-flag-CypA (S1LEK3-OE) or pCMV2-flag (S1LEK-Ve).

 
Cyclophilin A knockdown did not correlate with increased angiogenesis. To determine a potential mechanism to account for this difference in growth seen with CypA knockdown, we carried out a systematic analysis to look for involvement of CypA in key tumor biological pathways. One determinant of tumor growth is the ability of a nascent tumor to recruit a vasculature, or angiogenesis. Hypoxia is a primary stimulus for angiogenesis (14). To determine whether CypA is involved in the cellular response to hypoxia, CypA knockdown and control cells were treated in vitro with the hypoxia mimetic deferrioxamine, an iron chelator that stabilizes the HIF-1{alpha} transcription factor and enhances its DNA binding activity (15). No difference in cell viability was observed after deferrioxamine treatment at the standard 100 µmol/L dose (data not shown; ref. 16). Secretion of vascular endothelial growth factor (VEGF), a hypoxia-induced factor crucial for angiogenesis, was not detectable in CypA knockdown and control cells by Western blot (data not shown). To determine whether CypA knockdown directly affects angiogenesis in vivo, tumors were stained for the endothelial antigen CD31, and CD31-stained microvessels were quantitated (Fig. 4A and B). No significant difference in mean vessel density was observed between CypA knockdown and control tumors. CypA thus did not seem to play a role in angiogenesis in these tumors.



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  		Figure 4. CypA knockdown does not affect angiogenesis. A, CD31 immunohistochemistry in CypA knockdown (C4), scrambled (S5), and parental 5M2 tumors; B, microvessel density from three to four tumors per group. Values shown are the average microvessel density of the knockdown clones C1, C2, and C4 versus S5 and 5M2.

 
Cyclophilin A knockdown correlated with increased apoptosis. Serum withdrawal is a well-established in vitro model for inducing apoptosis in many cell types. To determine whether CypA expression contributes to increased cell viability following serum withdrawal, CypA knockdown (C1, C2, and C4) and control cells (S5 and 5M2) were grown in media without serum for 9 days. Obvious cellular detachment occurred, and greater cell death was observed in the knockdown cells after serum withdrawal compared with the control cells. However, statistical significance between knockdowns and both scrambled and parental controls was approached only at 9 days of serum withdrawal, and with knockdown clones C1 and C2 but not C4 (Fig. 5). To evaluate apoptosis in vivo, CypA knockdown and control xenografts were stained for terminal deoxyribonucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) reactivity. CypA knockdown tumors showed a significantly greater number of TUNEL-positive nuclei than scrambled (86.7 ± 25.1 versus 53.1 ± 8.98, P = 0.004) or parental tumors (86.7 ± 25.1 versus 43.6 ± 5.33, P = 0.0008; Fig. 6A and B). It has been shown that addition of recombinant CypA to cultured cardiac myocytes increases the antiapoptotic protein bcl-2 (17). To determine whether a correlation between CypA levels and bcl-2 exists in 5M2 cells, levels of the prosurvival protein bcl-2 in knockdown and control tumor xenograft lysates were compared by Western blot. No correlation with CypA knockdown was found (data not shown). Thus, CypA knockdown led to some sensitivity to serum starvation–induced cell death in vitro, and significantly increased apoptosis in vivo.



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  		Figure 5. CypA knockdown correlates with increased sensitivity to serum withdrawal. Cells at 20,000 per well were plated in replicates of four and serum was withdrawn for 9 days. Viable cells were counted at the indicated time points by Trypan blue exclusion.

 


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  		Figure 6. CypA knockdown correlates with increased apoptosis in vivo. A, TUNEL assay in CypA knockdown (C4), scrambled (S5), and parental tumors; B, average number of TUNEL-positive nuclei from three to four tumors per group. Values shown are the average number of TUNEL-positive nuclei of the knockdown clones C1, C2, and C4 versus S5 and 5M2.

 
Cyclophilin A knockdown correlated with decreased proliferation. Tumor growth is often considered a balance between apoptosis and proliferation. To determine whether CypA knockdown had an intrinsic effect on cell proliferation, [3H]thymidine incorporation was measured in knockdown and control cells. A difference was noted between knockdowns and parental (10,312 ± 3,201 versus 18,423 ± 2,067 cpm, P = 0.003) but not between knockdowns and scrambled control (10,312 ± 3,201 versus 14,777 ± 7,912 cpm, P = 0.433; Fig. 7). To test whether CypA knockdown had an effect on cell growth in culture, population doubling times were measured. No significant difference between C1, C2, C4, S5, or 5M2 parental was noted [population doubling times (hours): C1, (10.6); C2, (11.5); C4, (10.5); S5, (13.1); 5M2, (10.2)]. When CypA knockdown and control xenograft tumors were stained for the nuclear cell cycle antigen Ki67 (Fig. 8A), knockdown tumors showed significantly fewer Ki67-positive nuclei than parental (250.8 ± 53.6 versus 404 ± 41.2, P = 0.0006). The difference between knockdown and scrambled control was not significant (250.8 ± 53.6 versus 330.4 ± 86, P = 0.143; Fig. 8B). Thus, CypA knockdown led to decreased proliferation compared with controls in vitro and in vivo, but this trend was of borderline significance.



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  		Figure 7. CypA knockdown correlates with decreased proliferation in vitro, as assessed by [3H]thymidine incorporation (C1, C2, and C4, CypA knockdown; S5, scrambled; 5M2, parental).

 


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  		Figure 8. CypA knockdown correlates with decreased proliferation in vivo. A, Ki67 immunohistochemistry in CypA knockdown (C4), scrambled (S5), and parental tumors; B, average number of Ki67-positive nuclei from three to four tumors per group. Values shown are the average Ki67-positive nuclei of the knockdown clones C1, C2, and C4 versus S5 and 5M2.

 
Cyclophilin A knockdown correlated with decreased fluorodeoxyglucose uptake in vivo. To gain additional insight into the in vivo effect of CypA, tumor metabolism was assessed by [18F]fluorodeoxyglucose positron emission tomography ([18F]FDG-PET) imaging in the CypA knockdown and parental 5M2 tumors (Fig. 9). By FDG-PET, the CypA knockdown xenograft tumor had ~2-fold less FDG uptake than parental (standardized uptake value: 2.34 versus 4.05, respectively). FDG-PET has been shown to correlate with flow cytometric parameters of proliferation (percentage of S-phase nuclei, S + G2-M) in head and neck cancer (18). Moreover, the standardized uptake value, a normalized measure of FDG uptake, was found to be proportional to non–small-cell lung cancer tumor doubling times (19). Therefore, these results imply that CypA knockdown may be correlated with decreased proliferation and metabolism in vivo in these tumors.



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  		Figure 9. CypA knockdown is correlated with decreased tumor glucose metabolism in vivo. CypA knockdown (C1) or 5M2 parental xenograft–bearing mice were injected i.p. with 600 to 700 µCi [18F]FDG and emission scan data were acquired for 45 minutes using the MicroPET scanner at the Duke Center for In vivo Microscopy. Coronal images are shown demonstrating significant [18F]FDG uptake in the tumor. Images have been normalized for nonspecific background uptake.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Elucidation of novel molecular targets has enormous potential for improving cancer treatment, as recently evidenced by targeted therapy in patients with an epidermal growth factor receptor mutation in lung cancer (20–22). Previous reports from our laboratory showed overexpression of CypA in lung cancer. The current study is an extension of that work and attempts to show a role for CypA in tumor biology.

CypA is known to bind the T-lymphocyte tyrosine kinase Itk and regulates its signaling via prolyl isomerization, acting as a "molecular switch" (23). CypA also binds to the prolactin receptor and potentiates prolactin-induced downstream signaling via the Jak2/signal transducers and activators of transcription 5 pathway by prolonging Jak2 phosphorylation and inhibiting Rac activation (24). It is not known whether such a signaling mechanism exists for CypA with kinases in tumor cells. Pin1, also a peptidyl-prolyl isomerase, is specific for phosphotyrosyl-prolyl residues and interacts with mitotic kinases, but no such specificity has yet been shown for CypA.

The current study used RNA interference to stably inhibit expression of CypA in lung cancer cell lines, and showed that CypA knockdown significantly inhibited tumor growth in two lung cancer cell lines. Using a CypA overexpression construct with the S1LEK transformed small airway epithelial line, we further showed that increased CypA expression correlated with dramatically faster tumor formation in vivo.

To uncover pathways in which CypA might be acting, we proceeded with a systematic inquiry into the general areas of angiogenesis, proliferation, and apoptosis using the CypA knockdown cells. We found that CypA knockdown leads to decreased proliferation in vitro, as measured by [3H]thymidine incorporation, and in vivo, as quantitated by Ki67 immunohistochemistry. A correlation has been shown between FDG-PET and cell proliferation, and our imaging data support the Ki67 results that show an effect of CypA on proliferation in vivo. It was shown that CypA can interact with Rb in vitro and is required for Rb-mediated, retinoic acid–induced neuronal differentiation in p19 embryonal carcinoma cells, but an in vivo effect on tumor cell proliferation was not assessed in these studies (6, 25).

Malignant transformation is associated with increased metabolic demands, including increased glucose utilization, a decrease in glucose-6-phosphate, and an up-regulation of glucose transporters, and FDG-PET is traditionally viewed to reflect the degree of metabolic activity of a tumor (26). The decreased FDG uptake we observed in the CypA knockdown tumors reveals an effect on glucose metabolism, although further studies are needed to elucidate the exact mechanism.

We also showed that CypA knockdown tumors have a significantly greater degree of apoptosis than both scrambled and parental controls as measured by TUNEL assay. The absence of a correlation between CypA level and bcl-2 expression suggests that CypA acts in apoptotic signaling independent of bcl-2. We investigated the possibility that CypA may influence apoptosis by contributing to cellular resistance to apoptosis initiated by physiologic stressors. Such a stressor is serum withdrawal, which is known to cause apoptosis in many cell types, and may approximate the absence of growth factors as what a nascent tumor implant experiences before development of vasculature. We observed that after 9 days of serum withdrawal, CypA knockdown cells showed a significant reduction in the number of viable cells. These in vitro serum withdrawal data support an antiapoptotic role for CypA, as suggested by the increased TUNEL positivity seen in the CypA knockdown tumors in vivo.

Angiogenesis is also essential for tumor growth, and may be enhanced by increased endothelial cell survival and activation. In a recent report, recombinant CypA induced extracellular signal-regulated kinase, c-jun NH2-terminal kinase, and p38 kinase activation, as well as expression of adhesion molecules E-selectin and VCAM-1, in human umbilical vein endothelial cells (HUVEC; ref. 27). In another report, exogenous CypA increased HUVEC proliferation, migration, invasive capacity, and tubulogenesis (28). CypA, besides being an intracellular protein, has been shown to be secreted by lipopolysaccharide-activated macrophages and other cell types (27, 29, 30). We have found CypA to be secreted by 5M2 adenocarcinoma cells by Western blot of conditioned medium (data not shown), and these lines of evidence led us to hypothesize a possible paracrine effect of tumor cell–secreted CypA on endothelial cells, and hence on angiogenesis.

However, in the current study, no difference in angiogenesis was seen between CypA knockdown and control tumors as quantitated by CD31 staining and microvessel density. It is known that CypA is secreted by endothelial cells and vascular smooth muscle cells under conditions of oxidative stress and inflammation (30), and it is possible that such an autocrine effect of CypA on endothelial cells may have obscured the detection of a paracrine proangiogenic effect of tumor cell–derived CypA. Moreover, this study did not address the functional maturity of the tumor microvasculature, and it is possible that immunostaining with markers of perfusion and hypoxia (Hoechst dye and pimonidazole, respectively) may reveal a CypA-dependent difference in angiogenesis not shown by CD31 microvessel density.

Treatment of CypA knockdown and control cells with deferrioxamine in vitro to mimic hypoxia (15) did not reveal a difference in cell viability. In addition, we were not able to detect VEGF, a key proangiogenic mediator induced by hypoxia, in the culture medium by Western blot; hence, CypA-dependent differences in VEGF secretion were not assessed.

In summary, we have shown that knockdown of CypA overexpression in lung cancer cells leads to slower-growing and smaller tumors in severe combined immunodeficient mice. Our data suggest that CypA knockdown affects cellular proliferation and apoptosis. We further studied the effect of CypA using a CypA overexpression model in a transformed primary airway epithelial line, and found that it was correlated with an increased rate of tumor formation in vivo.

Given these data, it may be that inhibition of CypA will prove to be therapeutically useful. CsA is a well-known inhibitor of peptidyl-prolyl isomerase activity of CypA and has been studied in lung cancer, mainly in the context of chemotherapy regimens via its inhibition of the P-glycoprotein multidrug resistance protein (31–33). It has been shown to induce apoptosis in cancer cells, such as retinoblastoma and melanoma, but the exact mechanism has not been elucidated (34–36). CsA has several drawbacks as a therapeutic agent (i.e., through "off-target" effects—CsA is a potent immunosuppressant through inhibition of the calcineurin/nuclear factor of activated T-cell pathway in T cells, and by effects on transforming growth factor ß—CsA has been shown to cause tumor progression in non–small-cell lung cancer; ref. 37). Nonimmunosuppressive CsA derivatives exist (38), and treatment with such derivatives, or novel inhibitors of CypA, may lead to improvement in clinical outcomes for a certain subset of patients with lung cancer.


   Acknowledgments
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank the staff of the Duke Center for In vivo Microscopy, an NCRR/NCI National Resource (P41 05959, R24-CA92656), in the Duke Department of Radiology for assistance with the imaging. We also thank Shideng Bao, Xing Guo, and Chaoyu Ma of the Department of Pharmacology and Cancer Biology for their valuable advice.

Received 1/ 8/05. Revised 6/14/05. Accepted 7/15/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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