CD43 in the nucleus and cytoplasm of lung cancer is a potential therapeutic target

Authors


Abstract

CD43 is a transmembrane sialoglycoprotein. Normally the molecule is only produced by white blood cells where it regulates functions such as intercellular adhesion, intracellular signaling, apoptosis, migration and proliferation. Two CD43 antibodies were used to interrogate 66 cases of non-small cell lung cancer (NSCLC) and 24 cases of small cell lung cancer (SCLC). In addition, we engineered the CD43-positive lung cancer cell line A549 to stably express either non-targeted or CD43-targeted small-interfering RNA (siRNA). These lines were then subjected to in vitro assays of apoptosis, natural killer (NK) cell cytotoxicity, intercellular adhesion and transendothelial migration. A xenograft mouse model evaluated the ability of the lines to grow primary tumors in vivo. CD43 was found to be expressed in the majority of both SCLC and NSCLC. Inclusive of CD43-negative tumors, differential patterns of nuclear and cytoplasmic expression of CD43 define four molecular subcategories of lung cancer. Targeting CD43 in A549 lung cancer cells, increased homotypic adhesion, decreased heterotypic adhesion and transendothelial migration, increased susceptibility to apoptosis and increased vulnerability to lysis by NK cells. Furthermore, targeting inhibited the growth of primary tumors in nude mice.

Despite major advances in the field of medical oncology, lung cancer remains the leading cause of cancer death in the United States among both men and women. With an estimated 221,130 new cases and 156,940 deaths in 2011, lung cancer is responsible for more than 25% of all cancer deaths.1 Two studies have reported that lung cancer might be characterized by expression of the sialoglycoprotein CD43 normally only produced by leukocytes.2,3 The first study reported that 13 out of 13 cases of non-small cell lung cancer (NSCLC) expressed CD43 while two out of two cases of small cell lung cancer (SCLC) were CD43-negative.2 The second study reported that one out of three primary lung tumors exhibited CD43 expression.3 One of these three tumors was a small-cell carcinoma and the other two were squamous cell carcinomas. Here, we report the analysis of 90 cases of lung cancer using antibodies that recognize either the N or C terminus of CD43. In addition, we report the functional consequences of expressing CD43-targeted small-interfering RNA (siRNA) in the lung cancer cell line A549.

Material and Methods

Patient material

Paraffin-embedded formalin-fixed tissues representing 90 cases of lung cancer and one mild case of lung silicosis were provided free-of-charge by the Gundersen Foundation BioBank (http://www.gundluth.org/biobank). Each case was sectioned, stained with hematoxylin and eosin and the histological diagnosis verified. All experimental procedures were approved by the Human Subjects Committee of Gundersen Clinic, La Crosse, WI. Formalin-fixed, paraffin-embedded sections of normal human lung tissue were purchased from US Biomax (Rockville, MD) (Catalog number HuFPT131Aa).

Generation of the polyclonal antibody SSGZ

Covalab S.A.S. (Villeurbanne, France) synthesized a peptide of 26 amino acids with the sequence NH2-PLVASEDGAVDAPAPDEPEGGDGAAP-COOH. This corresponds to the terminal residues of the intracellular domain of CD43.4, 5 The same company then used glutaraldehyde crosslinking to conjugate the N-terminus of the peptide to keyhole limpet hemocyanin. Next, 0.5 ml containing 100 µg of the conjugated peptide was mixed with 0.5 ml of Complete Freund Adjuvant and this was then injected intradermally into a New Zealand White rabbit. After 21 days and then again after 42 days intradermal injection was repeated but with Incomplete Freund Adjuvant. After 63 days and then again at 91 days subcutaneous injection was performed with Incomplete Freund Adjuvant. After 116 days serum was drawn and IgG immunopurified.

Abbreviations

AAALAC-I: Association for Assessment and Accreditation of Laboratory Animal Care International; AF: attachment factor; BCECF-AM: 2′7′-bis(carboxyethyl)-5(6)-carboxyfluorescein pentaacetoxymethyl ester; FAM: carboxyfluorescein; FBS: fetal bovine serum; FMK: fluoromethyl ketone; HBSS: Hank's balanced salt solution; HMEC: human microvascular endothelial cells; MVGS: microvascular growth supplement; NK: natural killer; OD: optical density; PBS: phosphate buffered saline; PMA: phorbol 12-myristate 13-acetate; siRNA: small-interfering RNA; TNFα: tumor necrosis factor alpha; VAD: valine-alanine-aspartic acid.

Immunohistochemistry

Formalin-fixed paraffin-embedded blocks containing human lung tissue were serially sectioned at 4 µm and dried overnight on Colorfrost® Plus microscope slides (Thermo Fisher Scientific, Waltham, MA). Next, sample slides were deparaffinized by a 60-min incubation at 60°C followed by four changes of xylene, three changes of 100% ethanol, two changes of 95% ethanol and storage in tap water. One slide from each block was stained with Hematoxylin and Eosin Y. The remaining slides were subjected to a 20-min incubation at 90–100°C in the presence of Epitope Retrieval Solution, pH 9 (Dako North America, Carpinteria, CA). Next, the slides were rocked for 5 min at room temperature with tissue covered by the peroxidase blocking reagent of the EnVision + System-HRP (horseradish peroxidase) [3,3′-diaminobenzidine (DAB)] (Dako North America). A rocking incubation was then performed at room temperature for 30 min with Surfact-Amps® X-100 (Thermo Scientific, Waltham, MA). One slide from each block was rocked for 45 min at room temperature with either an IgG non-immune rabbit or mouse antibody (Epitomics, Burlingame, CA). One slide from each block was also incubated with either the rabbit polyclonal antibody SSGZ or the mouse monoclonal antibody L10. Serial rocking incubations were next performed at room temperature for 30 min with labeled polymer-HRP anti-rabbit or anti-mouse, twice for 5 min with wash buffer and 5 min with DAB + Chromogen (Dako North America). Counterstaining was accomplished by dipping the slides in Hematoxylin, rinsing with tap water, dipping in 1% glacial acetic acid, rinsing again in tap water and then dipping in 1% ammonium hydroxide. Rinsing in 100% ethanol then xylene dehydrated the tissue that was finally protected by glass coverslips mounted with Permount® (Thermo Fisher Scientific). A pathologist (JJA) certified by the American Board of Pathology and a histotechnician (SEC) certified by the American Society for Clinical Pathology independently scored the degree of L10 and SSGZ staining in the nucleus and cytoplasm on a scale of 0–3 (Supporting Information Tables 1 and 2).

Table 1. Molecular classification of lung cancer based on intracellular patterns of CD43 expression
inline image

Cell culture

The human lung cancer cell line A549 was obtained from the American Type Culture Collection (Manassas, VA, USA). The human T-lymphocytic cell line Jurkat was kindly provided by Thomas Wileman (University of East Anglia, Norwich, England). The growth medium for A549 and Jurkat was composed of RPMI1640, 10% vol/vol fetal bovine serum (FBS), 100 units/ml penicillin and 100 μg/ml streptomycin sulfate. The growth medium for A549 cells stably expressing siRNA was supplemented with 0.5 µg/ml of puromycin. Human microvascular endothelial cells (HMEC-1) were a kind gift of Sean P. Colgan (University of Colorado, Aurora, CO). The growth medium for this cell line was composed of Medium 131 containing 100 units/ml penicillin, 100 μg/ml streptomycin sulfate and microvascular growth supplement (MVGS) (Life Technologies Corp., Carlsbad, CA). In addition, surfaces on which HMEC-1 were grown were coated with attachment factor (AF) (Life Technologies, Corp.). The human NK cell line YT2C2-PR was kindly provided by Edgardo E. Carosella (Hôpital Saint-Louis, Paris, France). The growth medium for this cell line was the same as that for A549 and Jurkat, except that 20% vol/vol FBS was used.

Lentivirus expressing siRNA

Four types of SMARTvector™ shRNA lentivirus particles were purchased from Thermo Scientific Dharmacon (Lafayette, CO). The first lentivirus constitutively expressed siRNA that did not target any known human gene. The three additional lentiviruses expressed siRNA targeting CD43 mRNA at the coding sequence 5′-GTACACCACTTCAATAACA-3′ and at the 3′ non-coding sequences 5′-GGCAGTTGGTATTTCCCGA-3′ and 5′-AGAGCTGAGGATTTGGCGA-3′.

Generation of cell line pools stably expressing siRNA

The three CD43-targeted lentiviruses were mixed and used together to infect A549 cells. The lentivirus expressing siRNA targeting CD43 mRNA at the sequence 5′-AGAGCTGAGGATTTGGCGA-3′ was used to individually infect A549 as was the lentivirus expressing non-targeted control siRNA. Since each siRNA gene contained within the lentiviruses was linked to a puromycin resistance gene, resistance to this antibiotic was used to select for stable siRNA expression. Western blot analysis demonstrated that A549 cells selected after simultaneous infection with three CD43-targeted lentiviruses exhibited a significant reduction in CD43 protein expression compared to A549 selected after non-targeted infection (Supporting Information Fig. 1).

Figure 1.

CD43 is expressed by primary tumors of NSCLC and SCLC but not by non-malignant lung tissue. (a–e) Sections of 5 µm cut from formalin-fixed, paraffin-embedded tissue obtained from adenocarcinoma NSCLC. (f–j) Sections of SCLC. (k, m, o) Sections of non-malignant lung tissue obtained from a patient with mild silicosis. (l, n) Formalin-fixed, paraffin-embedded sections of normal human lung tissue purchased from US Biomax (Rockville, MD) (Catalog number HuFPT131Aa). Sections were stained with hematoxylin and eosin (H&E), a non-immune mouse IgG antibody (NI-M), a non-immune rabbit IgG antibody (NI-R), the mouse monoclonal antibody L10 or the rabbit polyclonal antibody SSGZ. Antibody binding was visualized using the EnVision™+ System-HRP (Dako North America). Counter-staining with hematoxylin visualized nuclei. Brown precipitates indicate L10 and SSGZ binding to CD43. The case of NSCLC depicted exemplifies the pattern of CD43 expression most commonly found in this histologic class. The pattern is designated CN to indicate a CD43 staining score of at least 1 both in the nucleus and cytoplasm. The case of SCLC depicted exemplifies the CD43 expression pattern most commonly exhibited by this histology. The pattern is designated NO to indicate a CD43 staining score of at least 1 only in the nucleus. Arrowed are leukocytes present in non-malignant tissue that exhibit membrane staining (LK).

Homotypic and heterotypic adhesion assays

Homotypic adhesion assays were performed using confluent monolayers of A549 expressing either non-targeted or CD43-targeted siRNA. Heterotypic adhesion assays utilized confluent monolayers of HMEC-1. Separate cultures of non-targeted A549 and CD43-targeted A549 were trypinized, washed in Hank's balanced salt solution (HBSS) and incubated for 30 min in a tissue culture incubator with HBSS containing 5 µM 2′7′-bis(carboxyethyl)-5(6)-carboxyfluorescein pentaacetoxymethyl ester (BCECF-AM) (Calbiochem®, EMD Chemicals, Gibbstown, NJ). The cells labeled with BCECF-AM were then washed three times in HBSS. Next, labeled non-targeted A549 cells were added to the confluent monolayers of unlabelled non-targeted A549 or HMEC-1 at a multiplicity of 0.1. In the same way, labeled CD43-targeted A549 were added to confluent monolayers of unlabeled CD43-targeted A549 or HMEC-1. Labeled cells were settled by centrifugation, incubated for 5 min then gently washed twice in HBSS. Fluorescence intensity was measured at excitation and emission wavelengths of 485 nm and 530 nm, respectively.

Migration and invasion assays

Transendothelial migration assays were performed using the BD Falcon™ HTS FluoroBlok™ 24-Multiwell Insert system (BD Biosciences, Bedford, MA). The wells of the top insert plate were treated with AF and a monolayer of HMEC-1 cells was established. The monolayer was then activated for 8 hr with 100 ng/ml of phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St Louis, MO). After activation, monolayers were washed twice with HMEC-1 medium free of MVGS. Suspensions of non-targeted and CD43-targeted A549 cells were labeled for 1 hr with A549 culture media containing 10 µg/ml DilC12(3) fluorescent dye (BD Biosciences). These cells were next centrifuged, resuspended in HMEC-1 medium free of MVGS and 5 × 104 added to each well of the top insert plate that contained an activated monolayer of HMEC-1. The wells of the bottom assay plate contained HMEC-1 medium complete with MVGS that bathed the lower surface of the wells of the top insert plate. Consequently, the lack of MVGS in the top wells and its presence in the bottom wells established a chemoattractant gradient. The completed culture system was then placed in a tissue culture incubator and the fluorescence intensity of the lower surface of the top wells was measured over time at excitation and emission wavelengths of 549 nm and 565 nm, respectively. Invasion assays were performed using the BD BioCoat™ Tumor Invasion System (BD Biosciences). The design concept of this system is the same as that used for the assay of transendothelial migration except that the monolayer of HMEC-1 is replaced with a uniform layer of BD Matrigel™ Basement Membrane Matrix (BD Biosciences). Suspensions of non-targeted and CD43-targeted A549 were washed twice in serum-free RPMI1630 and 2.5 × 104 added to each well of the top insert plate that contained the matrix. As a control, A549 cells were also added to wells of top insert plates that contained no matrix. In all experiments, the wells of the bottom assay plates contained RPMI1640 complete with 10% FBS to set up a chemoattractant gradient. The assembled culture system was incubated for 12 or 24 hr, the medium was then removed from the wells of the top insert plate and this transferred to a second bottom assay plate. Here each well contained 500 µl of HBSS supplemented with 4 µg/ml calcein AM (BD Biosciences). After 1 hr in a tissue culture incubator, the fluorescence intensity of the lower surface of the top wells was measured at excitation and emission wavelengths of 494 nm and 517 nm, respectively. Percentage invasion was calculated by multiplying by 100 the result of dividing the fluorescence intensity of matrix-coated wells with the intensity of equivalent non-coated wells.

Assay of apoptosis

Wells in 12-well flat-bottomed tissue culture plates were seeded with 5 × 104 non-targeted or CD43-targeted A549. After 24 hr, the culture media was removed and replaced with identical media except for the addition of 1.5 ng/ml of tumor necrosis factor α (TNFα) (Immunochemistry Technologies, LLC, Bloomington, MN). Cells were cultured in a tissue culture incubator for 36 hr then activated caspases detected using a FAM-FLICA™ in vitro Poly Caspases Assay Kit (Immunochemistry Technologies, LLC). Six independent microscope fields were imaged for each cell line both in white-light phase contrast and fluorescence at an excitation wavelength of 490 nm and an emission wavelength of 515 nm. The total number of cells in a given field was counted from the phase contrast image. The numbers of cells undergoing apoptosis were counted in the same field from the fluorescent image. The percentage of cells undergoing apoptosis in a given field was then calculated.

Assay of NK cell cytotoxicity

Wells in 12-well flat-bottomed tissue culture plates were seeded with 1 × 104 of either non-targeted A549 or CD43-targeted A549. Two days later the NK cell line YT2C2-PR was added at a multiplicity of 1, 5, 10, 15, 20 or 25. Plates were then centrifuged for 1 min at 1,000 g to effect uniform settling of YT2C2-PR. After 16 hr, supernatants were gently mixed and aspirated to remove non-adherent YT2C2-PR and A549 cells. Next, 0.7 ml of 0.25% Rose Bengal dye was added to each well and after 3 min the excess removed by two washes in phosphate buffered saline (PBS). Dye was released from intact, adherent cells by the addition of 0.8 ml of 50% ethanol and optical density (OD) measured at 570 nm. Percentage cytotoxicity was calculated using the formula 100 × (A − [BC])/D where A is OD of A549 cells after mixing to remove non-adherent cells, B is OD of experimental wells, C is OD of adherent YT2C2-PR cells and D is OD of A549 cells without mixing to remove those that are non-adherent.

Xenograft mouse model

Female mice that were 3–4 weeks old and of the strain Hsd:Athymic Nude-Foxn1nu were purchased from Harlan Laboratories (Indianapolis, IN). Cultures of non-targeted and CD43-targeted A549 were trypsinized, washed twice in growth medium then resuspended at a concentration of 2 × 107 cells per ml in sterile PBS. Next, each mouse in series was swabbed with 70% ethanol in an area on the dorsal right or left hind flank and 200 µl of an individual cell preparation was injected at this site. The maximum length of palpable tumors was measured in three dimensions at 7, 14, 21, 28, 31, 37, 42 and 53 days after injection. Using these dimensions, tumor volume was calculated as an ellipsoid. All animal use protocols were approved by the Institutional Animal Care and Use Committee of the University of Wisconsin, La Crosse.

Results

Both SCLC and NSCLC are characterized by intracellular expression of CD43

A rabbit was immunized and boosted with a synthetic peptide of 26 amino acids corresponding to residues 375–400 of the primary translation product of CD43 mRNA.4,5 These residues are located at the C-terminus of the intracellular domain of CD43. The IgG population of antibodies was purified from the serum of the immunized rabbit and named SSGZ. The L10 antibody binds the N terminus of CD43.6 The SSGZ and L10 antibodies were then used to analyze formalin-fixed paraffin-embedded non-malignant lung tissue, primary lung tumors removed from 24 patients with SCLC, 28 patients with carcinoid NSCLC, 18 patients with squamous NSCLC and 20 patients with NSCLC of the adenocarcinoma histological type (Fig. 1). No immunohistochemical signal was detected in the non-malignant control tissue except on the plasma membrane of leukocytes as would be expected for CD43 that is normally expressed. However, in SCLC, carcinoid NSCLC, squamous NSCLC and adenocarcinoma at least 10% of the malignant tissue was CD43-positive in 86%, 100%, 89% and 95% of the cases, respectively (Table 1). In contrast to membrane expression of CD43 in leukocytes, the expression of CD43 in both SCLC and NSCLC was exclusively intracellular. L10 staining occurred predominantly in the cytoplasm while SSGZ staining localized predominantly to the nucleus. Based on the pattern of intracellular expression of CD43 four classes of lung cancer can be defined (Table 1).

Expression of siRNA targeting CD43 increases homotypic adhesion of A549 lung cancer cells

Nuclear expression of CD43 in leukocytes protects against apoptosis, controls gene expression and likely drives proliferation.7,8 In addition, domains within the 123 amino acids at the C terminal end of the molecule directly bind protein kinases.9 Therefore, CD43 expressed in the nucleus and cytoplasm of lung cancer could facilitate disease pathogenesis directly by increasing cell survival and proliferation and indirectly by controlling intercellular adhesion through “inside-out” signaling.10

In order to determine whether CD43 functionally contributes to the pathogenesis of lung cancer, we targeted its expression in the lung cancer cell line A549.11 A pool of A549 was generated that stably expressed a mixture of three different CD43-targeted siRNAs. In addition, one control pool was generated that stably expressed a scrambled siRNA targeting no known human gene. Homotypic adhesion assays were performed on the A549 pool expressing CD43-targeted siRNA and on the A549 pool expressing control non-targeted siRNA. Comparison of these two pools demonstrated that targeting CD43 increases homotypic adhesion over two fold (Fig. 2a). This result indicates that CD43 inhibits the ability of lung cancer cells to bind to each other. Since CD43 is not expressed at the surface of lung cancer cells, its ability to inhibit homotypic adhesion appears to be mediated by an indirect mechanism.

Figure 2.

CD43-targeting increases homotypic adhesion but decreases heterotypic adhesion of A549. (a) Adhesion of BCECF-AM-labeled non-targeted and CD43-targeted A549 to respective confluent monolayers of unlabeled non-targeted and CD43-targeted A549 (n = 4). Error bars represent means ± SEM. The difference between adhesion of CD43-targeted A549 and non-targeted A549 was calculated by a paired one-tailed Student t test to have a significance level of p < 0.001. (b) Adhesion of BCECF-AM-labeled non-targeted and CD43-targeted A549 to confluent monolayers of unlabeled HMEC-1 activated with PMA (n = 5). Error bars represent means ± SEM. The difference between adhesion of CD43-targeted A549 and non-targeted A549 was calculated by the paired one-tailed Student t test to have a significance level of p < 0.001.

Targeting CD43 decreases heterotypic adhesion of A549 lung cancer cells to human microvascular endothelial cells

In addition to inhibiting binding of lung cancer cells to each other, CD43 could also facilitate tumor break-up by mediating the binding of lung cancer cells to the surrounding non-malignant endothelium. If this hypothesis is correct then CD43-targeting should decrease such heterotypic adhesion. Therefore, in order to test this hypothesis, we performed heterotypic adhesion assays between HMEC and either CD43-targeted or non-targeted A549 cells. The result of this analysis demonstrated that CD43 targeting decreased A549 adhesion to endothelial cells by 53% (Fig. 2b). Therefore, CD43 expression by A549 inhibits homotypic adhesion but positively mediates heterotypic adhesion.

Targeting CD43 decreases A549 transendothelial migration but not extracellular matrix invasion

Heterotypic adhesion represents an initial step in tumor invasion and metastasis. Therefore, since CD43-targeting reduced A549 adhesion to endothelial cells, we tested the hypothesis that such targeting would also inhibit the ability of A549 to migrate through an endothelial monolayer. Using a transendothelial migration assay, CD43-targeting was determined to reduce migration by an average of 50% over a period of 24–60 hr (Fig. 3a). Therefore, CD43 expression by A549 mediates not only static endothelial adhesion but also dynamic transendothelial migration. In contrast to migration through endothelial cells, invasion by A549 into a basement membrane extracelluar matrix was unaffected by CD43-targeting (Fig. 3b). Therefore, transendothelial migration mediated by CD43 would appear to involve integral components of endothelial cells rather than components of the extracellular matrix.

Figure 3.

CD43-targeting inhibits A549 transendothelial migration but not extracellular matrix invasion. (a) Transendothelial migration of DilC12(3)-labeled non-targeted and CD43-targeted A549 through monolayers of HMEC-1 activated with PMA (n = 9). Error bars represent means ± SEM. The difference between migration of CD43-targeted A549 and non-targeted A549 was calculated by a paired one-tailed Student t test to have a significance level of p < 0.001 for all time points. (b) Invasion of non-targeted and CD43-targeted A549 into BD Matrigel™ Basement Membrane Matrix (BD Biosciences) (n = 6). After invasion, A549 cells were labeled with calcein AM. Error bars represent means ± SEM. No significant difference in the invasive ability of non-targeted and CD43-targeted A549 was detected.

Targeting CD43 increases the sensitivity of A549 cells to apoptosis induced by TNFα

Exogenous over-expression of recombinant CD43 has been shown to protect the human colorectal cancer cell line SW480 from apoptosis.12 This observation suggests that endogenous expression of CD43 in A549 may also protect against apoptosis. In order to test this possibility we attempted to induce apoptosis of non-targeted and CD43-targeted A549 by a 36-h treatment with TNFα (Figs. 4a and 4b). Under these conditions non-targeted A549 exhibited no detectable early phase apoptosis, as assessed by the expression of activated caspases. However, in contrast, activated caspases were detected in 21% of CD43-targeted A549. Consequently, CD43 appears to protect A549 lung cancer cells from at least one anti-tumor response of the immune system.

Figure 4.

CD43-targeting increases the susceptibility of A549 to apoptosis and NK-mediated lysis. (a) Percentage of non-targeted and CD43-targeted A549 undergoing apoptosis after being treated for 36 hr with 1.5 ng/ml TNFα. Six independent microscope fields were analyzed for each cell line. In total, none of 887 non-targeted A549 cells exhibited apoptosis as measured by fluorescent detection of activated caspases. However, fluorescence was observed in 156 of 832 CD43-targeted A549. (b) Non-targeted and CD43-targeted A549 imaged in a microscope field either by white-light phase contrast or fluorescence. (c) The NK cell line YT2C2-PR was incubated with non-targeted A549 or CD43-targeted A549 at a multiplicity of 1, 5, 10, 15, 20 or 25. Percentage cytotoxicity effected by the NK cell line after 16 hr on each A549 cell line was calculated (n = 3). Error bars represent means ± SEM. The difference between lysis of CD43-targeted A549 and non-targeted A549 was calculated by paired one-tailed Student t tests to have significance levels of p = 0.022, 0.011, 0.027, 0.007, 0.020 and 0.102 for NK multiplicities of 1, 5, 10, 15, 20 and 25, respectively.

Targeting CD43 increases the susceptibility of A549 lung cancer cells to lysis by NK cells

Our analysis indicates that CD43 expressed by A549 protects against apoptosis induced by TNFα (Figs. 4a and 4b). The production of TNFα is a mechanism by which NK cells affect their cytotoxic function.13 Therefore, our studies suggest that CD43 expression could contribute to lung cancer pathogenesis by protecting malignant cells from NK attack. We tested this hypothesis by using non-targeted and CD43-targeted A549 cells in NK cytotoxicity assays. Over a range of NK cell multiplicity, A549 cells simultaneously expressing three CD43-targeted siRNAs exhibited higher susceptibility to lysis compared to non-targeted cells (Fig. 4c). The most striking effect occurred at a ratio of NK to A549 of 1:1. Here CD43-targeted A549 was 3.9-fold more susceptible to lysis compared to the non-targeted control. These data support the hypothesis that targeting CD43 in lung cancer cells increases their susceptibility to NK lysis and thus has the potential to limit lung cancer pathogenesis.

Targeting CD43 limits primary tumor growth in a xenograft mouse model of lung cancer

Our studies in vitro demonstrate that CD43 targeting increases the homotypic adhesion of lung cancer cells, decreases their adhesion to endothelial cells, reduces transendothelial migration and increases susceptibility to apoptosis and NK cytotoxicity. Taken together, these findings provide the basis for the hypothesis that CD43 targeting could inhibit lung cancer pathogenesis in vivo. This hypothesis was tested using a xenograft mouse model (Fig. 5a). When non-targeted A549 cells were injected subcutaneously into eleven Hsd:Athymic Nude-Foxn1nu mice all produced primary tumors. The average size of the tumors at 53 days was 381 mm3. In contrast, A549 cells stably expressing three CD43-targeted siRNAs produced tumors with an average size of 90 mm3 at 53 days. Furthermore, five of the eleven mice that were injected produced no detectable tumor at all. A549 cells expressing only one CD43-targeted siRNA produced tumors averaging 237 mm3 in each of nine injected mice. These results demonstrate that CD43-targeting limits in vivo the growth of primary lung tumors in a dose-dependent manner (Fig. 5b).

Figure 5.

CD43-targeting reduces primary tumor growth in vivo. A549 expressing non-targeted siRNA or one (×1) or three (×3) CD43-targeted siRNAs were injected subcutaneously into the dorsal right or left hind flank of mice of the strain Hsd:Athymic Nude-Foxn1nu (n = 11, 9 and 11, respectively). (a) The volume of palpable tumors measured 7, 14, 21, 28, 31, 37, 42 and 53 days after injection. Error bars represent means ± SEM. (b) The volume of palpable tumors measured 53 days after injection. Error bars represent means ± SEM. The difference in the volume of tumors produced by non-targeted A549 compared to A549 cells expressing one CD43-targeted siRNA was calculated by a paired one-tailed Student t test to have a significance level of p = 0.127. The difference in the volume of tumors produced by non-targeted A549 compared to cells expressing three CD43-targeted siRNAs had a significance level of p = 0.011. The difference in tumor volume produced by A549 cells expressing one compared to three CD43-targeted siRNAs had a significance level of p = 0.030.

Discussion

Under normal circumstances CD43, which has also been called large sialoglycoprotein, gpL115, leukosialin and sialophorin, is only expressed on the surface of leukocytes and platelets.9,14 The mature CD43 molecule is composed of 381 amino acids divided between a 235 residue extracellular region, a 23 residue transmembrane region and a 123 amino acid C-terminal intracellular region.4,5 The extracellular region contains approximately 84 sialylated O-linked carbohydrate units and appears by electron microscopy to be a rod-like structure extending 45 nm from the cell surface.15

CD43 has been described as a Janus molecule after the Roman god with two faces.14 This analogy reflects the finding that CD43 can perform diametrically opposite functions. First, depending upon how it is engaged at the cell surface, CD43 can either induce or protect against leukocyte apoptosis.16–20 Second, depending upon the status of leukocyte activation, CD43 can act either as an anti-adhesion barrier molecule or a pro-adhesion receptor.16,21–28

While leukocytes are at rest, the length, bulk, abundance and strong negative charge of CD43 combine to inhibit adhesion and maintain leukocytes in the circulation.21–24 During leukocyte activation, the surface expression of CD43 is reduced both by repression of the gene by which it is encoded and also by proteolytic cleavage of its extracellular domain.8,29–34 In addition, CD43 is excluded from foci of cell–cell contact and accumulates at the contracting uropod during polarization.23,35–38 Together, this down-regulation and redistribution facilitate intercellular interaction and migration effected by other leukocyte molecules such as the β2 integrins.35 In addition to mitigating of its anti-adhesive function, CD43 also plays a positive role in the performance of activated leukocytes. Changes in the glycosylation pattern of its extracellular domain allow CD43 to function as a pro-adhesive counter receptor for galectin-1, ICAM-1, E-selectin, sialoadhesin and MHC Class I molecules.16,23,25–28,37,39,40

While CD43 is normally restricted in its expression to leukocytes and platelets, aberrant expression has been consistently described in colon and salivary gland cancers.41–44 In addition, limited analysis suggested that NSCLC but not SCLC might also be characterized by CD43 expression.2,3 Using the anti-CD43 antibodies L10 and SSGZ together with extensive patient samples we have determined that CD43 is frequently expressed not only by NSCLC but also by SCLC. Two independent observers agreed that only 3 of 61 NSCLC cases were CD43-negative and only 3 of 22 SCLC cases. The discrepancy between our findings that SCLC is predominantly CD43-positive and the previous report of SCLC to be CD43-negative likely stems from the different number of cases analyzed in the two studies and the use of different antibodies.

Numerous gene expression profiles have been compiled for lung cancer.45 However, none has identified CD43 as being aberrantly expressed. This highlights an intrinsic limitation of the DNA microarray technique since abnormal CD43 expression in lung cancer would appear to be due to the abnormal induction of translation and/or protein stability as opposed to induced mRNA stability and/or transcription.

A striking feature of CD43 expression both in NSCLC and SCLC is that it is exclusively intracellular with no plasma membrane localization characteristic of leukocytes. The implication of this result is that the trafficking of CD43 in lung cancer is distinct from that in leukocytes. Another striking feature of the expression of CD43 in lung cancer is that the C-terminal epitopes recognized by SSGZ are expressed predominantly in the nucleus while the N-terminal epitope recognized by L10 is localized predominantly in the cytoplasm. This segregation of the C and N termini of CD43 to different intracellular compartments implies a proteolytic cleavage event that produces a C-terminal fragment containing the nuclear localization signal of the molecule and a N-terminal fragment with this signal missing.7

While NSCLC and SCLC are similar in terms of the frequency of CD43 expression, they are quite distinct in terms of how this expression is exhibited. In NSCLC, 54% of cases show CD43 expression both in the nucleus and the cytoplasm. This molecular category of lung cancer we designated CN signifying dual cytoplasmic and nuclear expression. In contrast, 73% of SCLC exhibits CD43 expression only in the nucleus. This molecular category we designated NO signifying nuclear only expression. In addition to CN and NO, lung cancer can be classified as either CO signifying cytoplasmic only expression or NR signifying the antibodies we used in our study were non-reactive. Whether the differential aggressiveness of NSCLC and SCLC is related to their bias toward different molecular categories defined by CD43 remains to be determined. Nevertheless, it is apparent that future work needs to establish whether “CD43 classification” has clinically relevant predictive power.

During leukocyte activation, the C-terminal domain of CD43 has been shown to be cleaved away from the rest of the molecule and translocate to the nucleus where it protects against apoptosis and may drive proliferation.7,8,46 Our results show that in lung cancer 70% of all cases exhibit constitutive nuclear expression of this C-terminal region. As with leukocytes, it appears that nuclear expression in lung cancer also protects against apoptosis since targeting of CD43 in the lung cancer cell line A549 induces caspase activation in response to TNFα.

The nuclear localization of the C-terminal domain of CD43 in lung cancer contrasts with colon cancer where it localizes predominantly to the cytoplasm.42 This indicates that in lung cancer the C-terminal domain of CD43 is linked to its bipartite nuclear localization signal while in colon cancer it is not. The significance of these implied differential cleavage events to the pathogenesis of lung cancer compared to colon cancer is unknown. However, it is tempting to speculate that nuclear localization of CD43 in lung cancer protects against apoptosis and changes gene expression as it does in leukocytes.7,8 Cytoplasmic localization may not drive such processes and, therefore, may help to explain the generally less aggressive nature of colon compared to lung cancer.1

A role for CD43 in driving the pathogenesis of lung cancer is suggested by extrapolating its anti-adhesion function in resting leukocytes and its pro-adhesion, migration and survival functions in activated leukocytes. If the activated leukocyte functions are projected to lung cancer, then CD43 could drive pathogenesis by facilitating metastasis and protecting against apoptosis. If the resting function of CD43 is projected, it has been previously suggested that this could facilitate an escape from immunosurveillance.3,47 In addition, the anti-adhesion function could help in turning a primary tumor into a loose cellular mass that sheds potentially metastatic neoplastic cells into the circulation.

In order to directly determine whether CD43 expression is a driver of lung cancer pathogenesis we targeted its expression in the lung cancer cell line A549. Such targeting increased homotypic adhesion supporting the hypothesis that CD43 facilitates the break up of primary tumors such that they shed potentially metastatic cells. In contrast to the induction of homotyptic adhesion, CD43 targeting decreased the heterotypic adhesion of A549 to endothelial cells. Therefore, it can be envisioned that both the anti-adhesion and the pro-adhesion functions of CD43 conspire to facilitate metastasis by, on the one hand, pushing the primary tumor apart from within and, on the other, pulling it apart from without by binding the surrounding endothelium. These adhesive functions must be mediated by an indirect “inside-out” mechanism since CD43 is expressed inside lung cancer cells not on the outside (Fig. 1). Such “inside-out” signaling is a means by which other molecules control intercellular adhesion.10 In the case of CD43, analogous “inside-out” signaling could be effected by nuclear expression of the C-terminus changing patterns of gene transcription as described in leukocytes.7 Alternatively, “inside-out” signaling could be effected by CD43 through its function as a docking platform for protein kinases.9

As well as mediating homotypic repulsion and heterotypic adhesion, our targeting studies demonstrate that CD43 also inhibits lung cancer apoptosis induced by TNFα. In leukocytes it has also been shown that CD43 can protect against apoptosis.8,18,19 However, in this context, CD43 protects against apoptosis mediated by Fas, growth factor withdrawal and Treg suppression but not against apoptosis induced by TNFα.8,18 Therefore, CD43 appears to protect against different apoptosis pathways in cancer cells compared to hematopoietic cells. Protection against apoptosis induced by TNFα would be particularly beneficial to cancer cells as this is one of the principal ways in which effectors of the immune system such as NK cells kill tumors.13 Consistent with this reasoning, we found that CD43-targeting increased the susceptibility of A549 to lysis mediated by NK cells.

CD43 drives leukocyte polarization and locomotion.36,38 These findings suggested to us that CD43 expressed by lung cancer cells might also mediate migration. Comparison of CD43-targeted and non-targeted A549 demonstrated that CD43-targeting reduces transendothelial migration by 50%. However, in contrast, CD43-targeting failed to reduce the invasion of A549 into a basement membrane matrix. This extracellular matrix preparation consisted of growth factors, laminin, collagen IV, heparan sulfate, proteoglycans and entactin/nidogen. Therefore, these components appear insufficient to effect CD43-mediated migration.

Taken together, our in vitro results indicate that CD43 could contribute to lung cancer pathogenesis in vivo in a variety of ways. These range from protecting against apoptosis and attack by NK cells to actively driving metastasis through mechanisms of anti-adhesion, pro-adhesion and migration. In order to test the role of CD43 in lung cancer pathogenesis in vivo we used a xenograft mouse model. Here we established that CD43-targeting limited, in a dose-dependent manner, the growth of subcutaneous A549 primary tumors. The intrinsic proliferation rate of A549 is unaffected by CD43-targeting (data not shown). Therefore, the reduction in primary tumor growth in vivo suggests increased vulnerability to the murine immune system. The mice used in our xenograft model were of the strain Hsd:Athymic Nude-Foxn1nu. This strain produces B-lymphocytes, NK and myeloid cells. Therefore, in vivo CD43-targeting probably makes A549 more susceptible to attack by one or more of these immune effectors.

Acknowledgements

Authors would like to thank Leah Morgan and Daniel Katzenberger for excellent technical assistance. They also thank Amy Cooper for animal husbandry advice and Dr. Douglas White, Dr. Ward Jones and Dr. Steven Callister for helpful discussions during the preparation of the manuscript. Authors thank Cheryl Gilster for searches of pathology files. Finally, authors would like to thank lung cancer survivor Susan Eber for her inspiration.

Grant sponsors: Gundersen Medical Foundation, Kabara Cancer Research Institute, La Crosse, Wisconsin.

Ancillary