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From the Laboratoire de Pathologie Cellulaire,*
INSERM
EMI 9924, CHRU Grenoble, Grenoble, France; the Laboratoire de
Physiologie Générale,
UMR CNRS
6558, Université de Poitiers, Poitiers, France; the Division of
Medical Oncology,
University of Colorado
Health Sciences Center, Denver, Colorado; and
IBMIG,§
ESA CNRS 6031, Université de
Poitiers, Poitiers, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Receptors for class 3 semaphorins, including SEMA3F, have been cloned.19,20 These include two related proteins: neuropilin-121,22 and neuropilin-2.23 Interestingly, neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor VEGF165,24 the most potent endothelial cell mitogen of five VEGF isoforms. In addition to neuropilin-1, neuropilin-2 was isolated in the expression cloning experiment in the search for VEGF165 receptor. Neuropilin-1 also binds additional proteins including a VEGF-like protein from orf virus,25 placental growth factor-2 (a VEGF family member),26 and putative cell adhesion ligands.19,27 Neuropilin-1 is widely expressed in adult human tissues including lung,24 and neuropilin-2 is expressed in mouse embryonic lung.23 During development of the rat embryo, neuropilin-2 and SEMA3F are expressed in complementary patterns,28 and SEMA3F binds with higher affinity to neuropilin-2.23 In the hippocampus, neuropilin-2 is the functional receptor for SEMA3F.28
Loss of chromosome 3p is a consistent event in human lung cancer. Frequent loss of heterozygosity (LOH) and the finding of a common homozygous deletion in three small-cell lung cancer (SCLC) cell lines suggested that 3p21.3 contains a tumor suppressor gene.29 In support of this, it was shown that an 80-kb genomic DNA fragment from this region suppressed tumorigenic growth of mouse A9 cells in vivo.30 This fragment was localized to a region of gene duplication containing two GTP-binding proteins (GNAI-2 and GNA-T) and two genes coding for SEMA3F and SEMA3B (previously known as H-SemaV).2,3,4 Based on sequence-tagged sites reported by Daly et al,31 along with available genomic sequence data covering a majority of the deletion region (http://genome.wustl.edu/gsc/human/chrom3.shtml), one could predict that SEMA3F was contained in the suppressing fragment. This was recently confirmed in abstract form (Naylor et al, abstract 433, American Society for Human Genetics, Denver CO, 1998) showing that SEMA3F was responsible for the inhibition of mouse A9 tumorigenesis.
However, despite the apparent involvement of SEMA3F in the mouse A9 system and homozygous deletions in three cell lines, other alterations of SEMA3F have not been reported. By Northern blots, SEMA3F was found to be expressed in most lung cancer cell lines3,4 but with marked differences in expression levels including 4/23 SCLCs and 3/16 non-small-cell lung carcinomas (NSCLCs) without SEMA3F transcripts.4 In no studies have SEMA3F protein levels been examined. To further elucidate the role of SEMA3F in lung cancer, we screened tumors for protein expression using a specific polyclonal antibody raised against a unique peptide.32 In addition, neuropilin-1 and -2 expression patterns were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) along with immunohistochemical analysis of VEGF in the same tumor samples. Although Sema3A (SemaIII) can effectively compete with VEGF for binding to neuropilin-1,28,33 whether similar competition exists between SEMA3F and VEGF165 and neuropilin-1 or/and neuropilin-2 is unknown. Our findings imply that this does occur. Last, indirect immunofluorescence with laser-scanning confocal microscopy was used to analyze the cellular distribution of endogenous SEMA3F in tumor cell lines.
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Materials and Methods |
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Ninety-five lung tumors were obtained at the time of tumor resection in NSCLC and carcinoids, in three large-cell neuroendocrine carcinomas (LCNEC), and in one SCLC. Tumor samples from seven LCNEC and nine SCLC were obtained by diagnostic lymph node resection. The tumors were classified according to the 1999 World Health Organization (WHO) histological classification of lung tumors34 as follows: 64 non-small-cell lung carcinomas (non-neuroendocrine tumors) comprising 29 squamous carcinomas and 35 adenocarcinomas; and 31 neuroendocrine tumors comprising 10 SCLCs, 10 LCNEC (another high grade neuroendocrine tumor) and 11 carcinoids (7 typical and 4 atypical) according to newly defined criteria (WHO 1999). According to TNM-derived stage classification, 37 of 64 patients with NSCLC were at stage I, 10 at stage II, 17 at stage III, and 1 at stage IV at the time of tumor resection. Nine of 10 carcinoid tumors were at stage I and one (atypical) at stage II. Among patients with LCNEC, 4 were at stage I, 4 at stage III, and 4 at stage IV. Six patients with SCLC were at stage III and 4 at stage IV. Tumor samples were quickly frozen for immunohistochemical and RT-PCR analysis in nitrogen liquid-cooled isopenthane. Other samples were used for histological classification.
Cell Lines
A series of 18 lung cancer cell lines including NCI-H661 (large-cell NSCLCs), NCI-H1450 and NCI-H740 (SCLC) were obtained from the Lung Cancer SPORE Tumor Bank Core of the University of Colorado Cancer Center. The SCLC cell line GLC20 has been described previously35 and was kindly provided by Dr. Charles Buys (University of Gronigen, The Netherlands). Human acute myeloid leukemia Kasumi-1 cell line,36 K562 cell line (from American Type Culture Collection (ATCC), Manassas, VA) in addition to the human cervical tumor cell line HeLa, were grown in RPMI-1640 containing 10% fetal calf serum. The human neuroblastoma cell line SK-N-SH (HTB-11), obtained from Dr. M. O. Jauberteau (University de Limoges, France) was grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% calf serum. The human lung adenocarcinoma cell line Calu-3 (ATCC: HTB-55) was grown in DMEM/F-12 with 10% calf serum.
RNA Expression
Total RNA was prepared with the SV Total RNA isolation kit (Promega, Madison, WI), and RT-PCR was performed with SuperscriptII reverse transcriptase (Life Technologies, Inc.) using the procedure supplied by the manufacturer. Alternatively spliced 915- and 1008-bp fragments from the SEMA3F cDNA were amplified by PCR with the 59H8 and 39G5 primers as previously described.2 A 154-bp fragment of neuropilin-2 cDNA was amplified with the following primers: 5'-AAGAGCGAAGAGACAACCAC-3' and 5'-CATACATCCAACCACAGGG-3' with an annealing temperature of 55°C. A 373-bp neuropilin-1 cDNA fragment was amplified with the primers 5'-GAAAGATAGCCCCTCCTCC-3' and 5'-CCACAGTAACGCCCAATG-3' with touch-down conditions starting from 64°C with a decrease of 1°C for every two cycles to the final temperature of 55°C (which was followed by 15 more cycles). The specificity of the PCR reaction was checked by sequencing the amplified fragment.
Quantitative PCR
We assessed levels of SEMA3F transcription relative to G3PDH in lung tumors by quantitative PCR carried out with the GeneAmp 5700 (PE Biosystems, Norwalk, CT) quantitative PCR system with syber-green chemistry. The cycle at which a particular sample reaches an arbitrary threshold fluorescence level (Ct) is indicative of input quantity of that template. Two different sets of primers were used to quantify SEMA3F cDNA: one set was SEMA3F-r 5'-GAG TCA GGG AAG GGC AAG-3' and SEMA3F-f 5'-CAA GTG TGC GGA AGA TGG-3' giving a 130-bp product and the other one was SEMA3F-7/21f 5'-AGC AGA CCC AGG ACG TGA G-3' and SEMA3F-7/21r 5'-AAG ACC ATG CGA ATA TCA GCC-3' giving a 114-bp product. G3PDH cDNA was amplified with primers G3PDH For 5'-TGC ACC ACC AAC TGC TTA GC-3' and G3PDH Rev 5'-GGC ATG GAC TGT GGT CAT GAG-3' giving a 87-bp product. The PCR was carried out in 50-µl reaction volumes consisting of 1x PCR SYBR Green 10X buffer, 0.25 µM primers, 200 µM each dNTP and 0.03 units/µl AmpliTaq Gold (PE). cDNA was amplified as follows: 50°C for 2 minutes, 95°C for 10 minutes followed by 40 cycles at 95°C x 15 sec, 60°C x 1 minute.
Immunohistochemical Analysis on Normal Lung and Tumors
SEMA3F immunostaining was performed with a rabbit polyclonal, affinity-purified, anti-SEMA3F antibody (3443–3AP) that was raised against a 16-amino acid peptide located in the C-terminal domain of the protein.32 Five µm thick frozen sections were fixed in cold acetone (-20°) for 10 minutes and allowed to dry at room temperature for 3 h. The anti-SEMA3F antibody was applied overnight at a 1:50 dilution. VEGF immunostaining was performed on paraffin and frozen sections from the same tumors. Endogenous peroxidase was quenched using 0.3% hydrogen peroxide in distilled water for 5 minutes, at room temperature, before incubation with anti-VEGF antibodies. Polyclonal anti-VEGF A20 (Santa Cruz Biotechnology, Santa Cruz, CA) (1:400 dilution on paraffin sections; 1:800 on frozen sections) and monoclonal antibody mAb 293 (R&D Systems, Oxon, UK) (1:25 dilution) were incubated overnight at 4°C. Slides were washed in phosphate-buffered saline (PBS) and then exposed to the secondary biotinylated donkey F(ab')2 anti-rabbit antibody (Ab) (1:1000 dilution; Jackson Laboratory, West Grove, PA) or anti-mouse Ab (1:500 dilution; Jackson Laboratory), for 1 h at room temperature. All slides were then washed in PBS and incubated with the streptavidin, biotin, and peroxidase complex (1:400 dilution; DAKO, Copenhagen, Netherlands) for 1 h at room temperature. The chromogenic substrate of peroxidase was a solution of 0.05% 3.3'-diaminobenzidine tetrahydrochloride, 0.03% H2O2, and 10 mM imidazole in 0.05 mol/L Tris buffer (pH 7.6). The slides were counterstained with Harris’ hematoxylin. Normal rabbit or mouse IgG at the same concentration as the primary antibodies served as a negative control. SEMA3F and VEGF immunostaining were scored, taking into account the percentage of positive cells (from 1 to 100%) and intensity of staining (from 1 to 3), as compared with internal controls. Internal controls for SEMA3F were normal bronchi and alveolar epithelial cells as described in Results and considered as intensity 3. Normal controls for VEGF were normal bronchi and smooth muscle cells, both were recorded as intensity 2. The total score was established by multiplying the percentage of stained cells by the intensity of staining, giving scores from 0 to 300. Final scores of 0, 1, 2, and 3, were given for total scores of 0, 10 to 30, 30 to 150, and 150 to 300, respectively. Scores were assessed by two independent observers (EB and JR), and a consensus was obtained.
To assess the specificity of the polyclonal antibodies, we performed a blocking experiment with both anti-VEGF A20 and anti-SEMA3F antibodies by incubating the primary antibody with a 10 times excess weight of immunizing peptide: VEGF A20 blocking peptide (Santa Cruz) and the 16-amino acid SEMA3F peptide,32 respectively, at room temperature for 2 h before incubation on sections. The remainder of the incubation and detection were performed as above. No immunostaining with these preabsorbed antibodies was obtained.
Immunostaining and Confocal Microscopy on Cell Lines
Cells in culture were fixed in 1% paraformaldehyde in Tris-buffered saline solution (TBS: 20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 2 mM EGTA, 2 mM MgCl2) for 10 minutes and permeabilized during 6 minutes in cold methanol for immunostaining of endogenous SEMA3F. For double-staining of SEMA3F and F-actin, cells were fixed in 1% paraformaldehyde in TBS for 10 minutes and permeabilized for 5 minutes in 0.1% Triton X-100/TBS. F-actin intracellular organization was visualized by staining with 1 µg/ml tetramethylrhodamine B isothiocyanate (TRITC)-conjugated phalloidin. Immunostaining for SEMA3F was performed with the anti-SEMA3F antibody 3443–3AP32 diluted at 1:50, combined with a fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit immunoglobulin secondary antibody, diluted at 1:200. All antibodies were diluted in TBS containing 1% bovine serum albumin (BSA) and 2% goat serum. Negative controls were performed by preabsorbing the antibody with the immunizing peptide.32 Stained samples were mounted and viewed with x63 oil immersion objectives using a Zeiss epifluorescence microscope (Axiovert 135 mol/L) equipped with HBO 50 W mercury short-arc lamp. The fine distribution of SEMA3F and actin was analyzed by means of laser-scanning confocal microscopy (Biorad 1024) with an inverted microscope (Olympus IX70) equipped with x63 and x100 Neofluar oil immersion objectives. Samples were excited with the visible lines 488 or 568 nm of an argon/krypton laser beam for green and red fluorescence, respectively. Fluorescence emissions were collected via two photomultiplier tubes through a band pass filter (522DF35) for green FITC fluorescence, or a band pass filter (605DF32) for red TRITC fluorescence.
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Results |
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SEMA3F was detected with a polyclonal antibody raised against a 16-amino acid peptide as previously described.32 This affinity-purified antibody is useful for immunochemistry but does not work for immunoprecipitation and Western blot. The specificity was demonstrated by the following observations. First, in three separate lung cancer cell lines with homozygous deletions involving SEMA3F, no signals were observed. In contrast, in all other cell lines where SEMA3F mRNA was detected, the immunohistochemical stain was positive. Second, blocking with the immunizing peptide eliminated the signal. Third, the antibody recognized the wild-type protein in transfected insect cells, whereas no signal was observed in untransfected cells. Last, in support of these observations the immunizing peptide is a unique epitope among semaphorins as determined from an analysis of sequences present in GenBank.
Normal Lung Structures
In normal lung, SEMA3F was detected in all epithelial cells
(Figure 1A and B)
. The use of frozen
section (instead of paraffin section) does not allow expansion of lung
parenchyma; thus, it gives a false impression of interstitium
thickening (Figure 1B)
. In large bronchi (cartilaginous bronchi), basal
bronchial cells were stained more intensely than ciliated or goblet
cells. There was a stronger membrane staining in addition to mild
diffuse cytoplasmic staining. Mucous and serous gland cells displayed
the same staining pattern. In bronchioles, SEMA3F was essentially
restricted to basal epithelial cells with mild cytoplasmic and strong
membrane staining. All types of alveolar lining cells displayed only a
membranous staining pattern that was restricted to the apical
intra-alveolar surface except in areas of type II cell hyperplasia
where a mild cytoplasmic staining was observed in addition to
membranous staining. This pattern of staining resembled that observed
in basal bronchial and bronchiolar cells. Endothelial cells of the
alveolar capillary bed did not express SEMA3F, whereas about 20% of
vessels more than 100-µm in diameter (arterioles and venules)
displayed endothelial staining with the typical apical membrane
pattern. There was no apparent staining in lymphatic vessels, however
the lymphatic bed is quite obscured in normal lung.
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Before performing SEMA3F immunodetection, we first verified by RT-PCR that SEMA3F mRNA was expressed in 18 lung tumors. We did not have enough material to perform Northern blots. The two alternative spliced forms of SEMA3F cDNA described before2 were present in all samples. In addition, neuropilin-1 and neuropilin-2 mRNAs were expressed in the same samples. Although contamination by normal cells could have explained these findings, we found that a variety of 16 lung tumor cell lines expressed SEMA3F and neuropilin mRNAs by themselves. The GLC20 SCLC cell line used as a negative control for SEMA3F expression did not express SEMA3F as expected but was positive for neuropilin-2.
A total of 95 lung carcinomas, histologically classified according to
1981 and 1999 WHO criteria, were studied for SEMA3F immunostaining on
frozen sections. These included 29 squamous cell carcinomas, 35
adenocarcinomas, 20 high grade neuroendocrine (NE) lung tumors
consisting of 10 SCLC and 10 LCNEC,37
and 11 carcinoids (8
typical and 3 atypical). Because surgical samples were used in this
study, there was a higher number of low stage tumors (stage I to II: 47
cases) than high stage tumors (stage III to IV: 17 cases). Low grade NE
tumors (carcinoids) as well as high grade NE tumors (SCLC and LCNEC)
were included in attempt to correlate differences in SEMA3F levels with
tumor aggressiveness. The results are summarized in Table 1
.
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, respectively). Fewer
adenocarcinomas were negative or expressed low levels of SEMA3F (5/35:
14%), whereas the majority (86%) expressed intermediate or high
levels (scores 2 to 3) (Figure 1G)
. Thus, in NSCLC tumors overall,
16/64 (25%) had SEMA3F scores of 0 to 1 compared with staining of
normal lung epithelial cells (score = 3). In NE lung tumors, 50%
of high grade tumors (SCLC and LCNEC) showed an absence or low level of
SEMA3F expression in contrast to carcinoid tumors, which all exhibited
intermediate or high expression levels (P =
0.005). Overall, when all histological types were considered, 27% of
tumors displayed no or low SEMA3F, and 38 and 35% showed intermediate
and high levels, respectively.
We then examined SEMA3F localization according to histological NSCLC
subtypes. Membrane localization of SEMA3F was infrequent in squamous
cell carcinoms (6/27: 22%). However, when membrane staining was
observed, it gave an intercellular mosaic pattern reminiscent of
desmosomal junction, and it was often prominent at the basally oriented
membrane toward the tumor stroma consistent with the possible
involvement of SEMA3F in cell-cell adhesion or migration (Figure 1I)
.
In adenocarcinomas, most acinar and solid types (11/15) displayed
diffuse cytoplasmic staining, whereas 4 of 15 showed the basally
oriented pattern observed in squamous carcinomas. In contrast, 8/17
well differentiated adenocarcinomas of bronchioloalveolar and papillary
types displayed the characteristic membrane pattern of apical or
luminal membrane staining observed in their candidate normal progenitor
cells, ie, type II alveolar and Clara cells. In some NSCLC tumors,
small clusters of cells disseminated in the stroma expressed higher
levels of SEMA3F than cells in large lobules (Figure 1J)
. In high grade
NE tumors, SCLC and LCNEC never displayed membranous staining, which
was in marked contrast to that observed in carcinoid neoplasms.
Neovasculature in tumor stroma were essentially unstained except in 12 of 95 tumors in which 10 to 50% of the vessels were SEMA3F positive (2 squamous cell carcinomas, 3 adenocarcinoms, 2 LCNEC, 1 SCLC, 4 carcinoids). Lymphatic vessels, which appeared more developed and dilated than in the normal lung, were likewise unstained. However since fibroblasts were stained in approximately one-half of the tumors, we cannot exclude that some SEMA3F-positive fusiform cells in the stroma were actually endothelial cells lining obscured lymphatic channels.
SEMA3F Scores Correlate Inversely with Tumor Stage and Clinical Aggressiveness
In NSCLC, the TNM stage (comprised of tumor size, node status,
presence of metastasis) is presently the most useful means of
predicting prognosis. SEMA3F levels were evaluated in individual tumors
and compared with their stage (Table 2)
.
An intermediate or high level of SEMA3F (scores 2 to 3) was observed in
87% of limited stages NSCLCs (stage I to II) compared with 41% in
tumors of higher stages (III to IV) (P =
0.0005). Thus, in NSCLC, high SEMA3F scores correlated with low stage.
In addition, membrane staining correlated with low stages when all
tumor types were considered (P = 0.05). In NE
tumors, clinical aggressiveness and survival are highly dependent on
histological type as recently described in a large survival analysis of
200 cases.37
We observed that 50% (10/20) of high grade
NE tumors expressed no or low levels of H.SemaIV, whereas all
carcinoids (typical and atypical) expressed high levels (Table 1
and
Figure 1, K and L
). Thus, high SEMA3F scores were significantly more
frequent in low or intermediate grade NE tumors than in high grade
tumors.
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The results of quantitative VEGF immunostaining are presented in
Table 3
and examples are given in Figure 1
(D, F, H). Both anti-VEGF antibodies (polyclonal A20 and monoclonal
mAb 293) gave concordant results in 90% of tumors, and the highest
score was retained in the 10% cases with discordant immunostaining.
Overall, only 18/95 tumors (19%) showed no or low VEGF immunostaining,
whereas 29 and 41% exhibited intermediate or high levels,
respectively. When the mean VEGF score was chosen as a threshold value,
63% of NSCLCs displayed high VEGF levels. VEGF immunostaining was
cytoplasmic and diffuse on paraffin sections, but distinct membrane
staining was observed on frozen sections in all cases of NSCLC
expressing VEGF and normal lung. Surprisingly, however, most high grade
NE tumors and one-half of the carcinoids did not display membranous
localization despite high VEGF levels. No significant correlation could
be established between the intensities of SEMA3F and VEGF
immunostaining using the 0 to 3 scoring values (exact Fisher test
P = 0.6 and Spearman test P = 0.7).
However, when the localization of SEMA3F (membranous or only
cytoplasmic) was analyzed in relation with VEGF (Table 4)
, a significant correlation was found
between scores for VEGF immunostaining less than the mean value and the
membrane localization of SEMA3F. We noted that all bronchioloalveolar
and papillary adenocarcinomas (well differentiated types) lacking a
membranous localization for SEMA3F exhibited high levels of VEGF (more
than the mean value). In small clusters of cells disseminated in NSCLC
tumor stroma and expressing higher levels of SEMA3F than cells in large
lobules, VEGF staining was also enhanced (data not shown).
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As SEMA3F protein levels were found variable in lung tumors, one
hypothesis was that SEMA3F transcription efficiency would be
responsible for these differences. To investigate this, we assessed
levels of SEMA3F mRNA in selected tumors using quantitative
real-time RT-PCR (Figure 2)
. Compared to
normal lung, the levels of SEMA3F mRNA in tumors ranged from
68% to as low as 8% (Table 5)
. The
tumor containing the lowest level of SEMA3F mRNA (8%) was
negative for SEMA3F protein expression by immunochemistry. However,
only a loose correlation existed between SEMA3F mRNA levels
and SEMA3F protein scores. In addition to competition by
VEGF165 for binding to a common receptor
(discussed below), another variable could be the amount of
contaminating stromal cells in the tumor that ranged from 10 to 30%
(Table 5)
.
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Indirect immunofluorescence in conjunction with laser-scanning
confocal microscopy was used to analyze the cellular distribution of
endogenous SEMA3F in four malignant cell lines: NCI-H661 (large-cell
NSCLC), Calu-3 (lung epithelial adenocarcinoma), HeLa (cervical
carcinoma), and SK-N-SH (neuroblastoma). Paraformaldehyde- and
methanol-fixed cells displayed a granular cytoplasmic fluorescence
signal as shown by confocal images obtained in horizontal (Figure 3)
and vertical sections (not shown). No
signal was detected in nuclei of the four cell lines and in peripheral
vesicles in NCI-H661 cells (Figure 3, A and B)
. The SCLC cell line,
GLC20, used as a negative control, displayed a negligible nonspecific
fluorescence signal (not shown). Similarly, no signal was detected in
NCI-H661, Calu-3, HeLa, and SK-N-SH cells when the anti-SEMA3F antibody
was preabsorbed with the immunizing peptide.
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: arrows). These dense intercellular structures suggest the
possibility that SEMA3F could be associated with cell adhesion domains
reminiscent of adherens junctions. However, SEMA3F was not observed at
the basal membrane of Calu-3 cells that are also growing in islets. On
the contrary, SEMA3F was concentrated at the apical domain of polarized
cells (Figure 3C)
. This localization is reminiscent of that observed in
lung epithelia sections.
Cells with locomotion activity, like HeLa (Figure 3, D, E, G, and H)
and SK-N-SH (Figure 3F)
cells, migrated by forming a large
lamellipodium in the direction of the movement. In cells displaying
this morphological aspect, a spatial concentration of fluorescence was
observed in motile regions like leading edges or ruffling membranes at
the margin of lamellipodia or cellular protrusions (Figure 3, D
-F).
Arbitrary representations of the fluorescence intensity by color scale,
showed that SEMA3F staining is sometimes more intense at the edge of
all protrusions that in other parts of the cytoplasm (Figure 3, E and F)
. When F-actin was labeled in the same cells with FITC-conjugated
phalloidin, SEMA3F colocalized with actin microfilaments in ruffling
regions but was neither associated with ventral stress fibers or
cortical ring of microfilaments (Figure 3, G and H)
. Migrating cells
showed a prominent actin-rich region at the edge of lamellipodium,
followed by the lamella, a transition zone with low actin content
(Figure 3H)
. SEMA3F showed the same kind of distribution in this motile
region as in actin-rich membrane ruffles at the edge of cell
protrusions. This suggests a role of SEMA3F in cell motility.
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The mechanism and specificity of altered SEMA3F localization is presently unknown. We observed, by immunohistochemistry on frozen sections, that other membrane proteins (EGF receptor, IGF-1 receptors, NCAM molecules)40 were appropriately positioned in these tumors excluding the possibility that fixation conditions or other artifacts were responsible. Defects in the secretion of extracellular proteins are frequently observed in cancer cells41 (and references therein). Whether SEMA3F post-translational modifications such as cleavage by furin,42,43 N-glycosylation or homodimer formation43,44 can affect its localization is unknown. Another explanation could be that SEMA3F is unable to bind its receptor either because of a component deficiency or because of a competitor. We found by RT-PCR that neuropilin-1 and -2, the SEMA3F receptors, are expressed in all lung tumors and tumor cell lines. SEMA3F binds neuropilin-2 in vitro with higher affinity than neuropilin-1, kd ~ 0.1 and 1 nM, respectively,23 although the respective concentrations of these proteins in normal lung and tumors are unknown. Interestingly, lung cancer cells and especially poorly differentiated adenocarcinomas that had lost membrane staining of SEMA3F showed high levels of VEGF. Because neuropilin-1 and possibly neuropilin-2 are common receptors for both VEGF165 and SemaIII,24 VEGF might be a competitor for SEMA3F binding. This hypothesis is in agreement with other data showing that Sema3A (SemaIII) and SEMA3A (Collapsin-1) compete with VEGF for binding to neuropilin-1.28,33 On frozen sections of NSCLCs, we found VEGF localized at the plasma membrane, which is consistent with this possibility. It is thus tempting to attribute a function of SEMA3F in angiogenesis, because recent studies demonstrated that exogenous SEMA3A inhibited the capillary sprouting of endothelial cells from rat aortic ring segments.33 However, few vessels of median caliber are positive for SEMA3F, and lymphatic vessels in tumors are negative. This is in contrast with VEGF staining of stroma capillaries in the vast majority of tumors. Moreover, our observation that neither SEMA3F nor VEGF exhibited membrane localization in most high grade NE tumors renders this simple competition model more elusive and favors the possibility that one of the receptor components might be limiting. This is conceivable, because there is evidence that neuropilins form a complex with a promiscuous accessory molecule necessary for signal transduction10,28,45-47 recently identified as plexin.48,49 Alternatively, high turnover of the receptor with rapid internalization in these tumors has not been ruled out.
Immunolocalization of SEMA3F by confocal microscopy in Calu-3 cells (lung epithelial adenocarcinoma), confirmed that the protein is predominantly distributed in the apical domain of polarized cells, as for normal lung epithelia sections. However, in a large proportion of NCI-H661 cells (large cell NSCLC), showing no or little locomotion activity, SEMA3F was at the interface of adjacent cells reminiscent of adherens junctions. Also, in 22% of squamous cell carcinomas, SEMA3F was localized at cell junction interfaces and at the basally oriented membrane toward the tumor stroma supporting the hypothesis of SEMA3F involvement in cell adhesion or migration. Cell-cell adhesion through homotypic cadherin binding, as well as mobilization of actin-containing undercoats at adherens junctions, are inversely correlated with invasiveness and tumorigenicity.50 Disruption of this system is frequent in cancer. For example, E-cadherin-catenin complexes were delocalized to the cytoplasm in invasive clusters of 32 out of 44 primary lung tumors.51
In contrast, in more motile cell lines (HeLa and SK-N-SH), SEMA3F was
localized in both the cytoplasm and in motile regions such as leading
edges or ruffling membranes of lamellipodes or cellular protrusions
where actin microfilaments are colocalized. Likewise in NSCLC tumors,
small clusters of scattered cells presumably emanating from large tumor
lobules showed enhanced cytoplasmic SEMA3F expression. These two
observations suggest that SEMA3F could be involved in cell migration.
Actin-rich membrane ruffles and lamellipodia are known to be involved
in the dynamic actin reorganization necessary for extension of cell
protrusions and cell motility,52
and semaphorins are
believed to induce a net loss of F-actin in the leading edge of growth
cones53,54
rather than inducing microtubule polymerization
or depolymerization. These observations are relevant, because motility
factors are not only involved in physiological events like
embryogenesis or wound healing, but also in pathological processes such
as tumor invasion and metastasis. A recent study55
demonstrated the involvement of SEMA3A in neuronal crest migration in
the chick and in morphological changes induced to crest cells in
vitro after exposure to SEMA3A suggesting that this semaphorin
might operate to control the movement of cells. Similarly, other
results that relate to neuropilin function indirectly involve
semaphorins in migration. Neuropilin-1 is also a placental growth
factor-2 receptor,26
which induces migration of
endothelial cells. Neuropilin-1 expression, like VEGFR-2, is
up-regulated by tumor necrosis factor-
,56
which
enhances migration and wound healing triggered by
VEGF165.
The localization of SEMA3F at motile regions in HeLa and SK-N-SH cells seems, at first, contrary to the findings in NCI-H661 cells where SEMA3F was detected at the interface of adjacent cells. However, SEMA3F could be involved with membrane-associated proteins that regulate both adhesion and motility. It clearly appears that SEMA3F localization is highly dependent on the cell type, the tumor class, and/or the cell phenotype. These observations suggest that the cell transformation process could affect one or several partners involved in SEMA3F compartmentalization.
Our results support the involvement of SEMA3F in the pathogenesis of human lung cancer and its possible role in VEGF pathway. We also suggest that SEMA3F may be involved in cell-cell adhesion or migration and that SEMA3F distribution may be affected by changes in cell activity. We hypothesize that the reduction of SEMA3F expression in tumors (manifested by the loss of SEMA3F membrane staining) facilitates VEGF receptor activation and an enhanced migratory response to VEGF in part through the permeabilization of stroma extracellular matrix during this migratory process.57 Either of these potential pathways might be sufficient to explain how reduced expression of SEMA3F or, conversely, its overexpression in mouse A9 cells could lead to altered tumorigenicity. Whether other human semaphorins will function in a similar manner remains to be determined. For example, SEMA4D (CD100) a leukocyte transmembrane class 4 semaphorin,58 which induces B cells to aggregate and would improve their viability in vitro,59 is lost in follicular B cell lymphomas.60 Another example includes a semaphorin related to SEMA4D located at a chromosomal region, 5q21–22, which is frequently deleted in advanced lung cancers.61 The role of semaphorins in cancer is not limited to loss of expression, however. For example, expression of another member of the class 3 family, Sema3E (M.semaH), correlates with the metastatic ability of mouse tumor cells.62 Likewise, Yamada et al63 identified SEMA3C (H-sema E) as a non-multidrug-resistance gene in human cancers. This secreted semaphorin was found to be overexpressed in cis-platinum-resistant cell lines and was induced by diverse agents including X-ray and UV irradiation. Thus, the investigation of these molecules is likely to be very informative even outside the context of neurodevelopment.
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Acknowledgements |
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Footnotes |
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Supported by La Ligue Nationale Contre le Cancer (E. B.), CNRS UMR 6558 (B. C.), grant P30 CA 46934 and University of Colorado Lung Cancer SPORE CA5187–07 (H. D.), the generous equipment support from the Lulu Frankel Foundation (H. D.), the Association pour la Recherche sur le Cancer (E. B., J. R.), La Ligue Nationale Contre le Cancer, Comité de la Vienne (J. R.) and NATO (J. R., H. D.).
Accepted for publication November 29, 1999.
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