(Received for publication, September 28, 1995; and in revised form, March 1, 1996)
From the
The insulin-like growth factors (IGFs), IGF-I and IGF-II, are
potent mitogens for human lung and other epithelial cancer cell lines.
Previous studies in defined medium lacking added IGF or insulin suggest
that an IGF-related ligand can act as an autocrine growth factor for
many cancer cell lines through action via the type I IGF receptor
(IGF-R). Analysis of RNA isolated from human lung and breast cancer
cell lines by reverse transcription of mRNA and polymerase chain
reaction reveal that IGF-I and IGF-II mRNAs were co-expressed with
IGF-R in the majority of cell lines. IGF-I mRNA was detected in 11/12
small cell lung cancer cell lines (SCLC), 13/14 non-small cell lung
cancer (NSCLC) cell lines, and 1/2 breast cancer cell lines. IGF-II
mRNA was detected in 8/10 SCLC, 11/12 NSCLC cell lines, and 2/2 breast
lines. All cell lines expressed IGF-R. For analysis of IGF peptide
secretion, cell lines were adapted to growth in serum/hormone-free
culture medium (R), and to avoid interference by
IGF-binding proteins, secreted IGF peptides were isolated under acidic
conditions and analyzed by Western blotting. Based upon measurement of
the sensitivity of the anti-IGF antibodies for detection of recombinant
human IGFs, IGF peptides accumulated in conditioned medium at greater
than picomolar concentrations should have been readily detected. In
three cell lines (two lung and one breast) secreted IGF
immunoreactivity was detected as three molecular mass species of 23,
14, and 6 kDa. Isolation and NH
-terminal sequencing of each
of these species definitively identified them as differentially
processed forms of the IGF-II prohormone. Despite the high frequency of
IGF-I gene expression detected by reverse transcription-polymerase
chain reaction analysis, only one lung cancer cell line, NCI-N417d, was
found that unequivocally secreted IGF-I peptide. This direct sequence
determination unambiguously identifies IGF-II as the predominant IGF
involved in the autocrine growth stimulation of human lung and breast
epithelial tumor cell lines and supports a growing body of literature
that implicates IGF-II/IGF-R autocrine loops as a common growth
mechanism in epithelial carcinogenesis.
Recent studies have identified the type I insulin-like growth
factor receptor (IGF-R) ()as a potential control point for
transformed cells(1, 2, 3) . Using a variety
of approaches, it has been demonstrated that the growth and
tumorigenicity of transformed cells can be inhibited by the
perturbation of IGF-R function(1, 4, 5) , and
IGF-R has been implicated in the protection of tumor cells from
apoptosis(3) . Human epithelial cancer cell lines express the
IGF-R(6, 7, 8, 9) , proliferate in
response to the IGFs(4, 6, 8) , and exhibit
reduced rates of growth when cultured in the presence of the monoclonal
antibody
-IR3, which inhibits the interaction of the IGF-R with
its ligands(4, 8, 10) . The ligands that bind
IGF-R with high affinity are the insulin-like growth factors I (IGF-I)
and II (IGF-II). These studies show that in vitro in defined
medium that lacks IGFs or insulin, an IGF-related ligand can function
as an autocrine growth factor for human epithelial cancer cell lines.
Therefore, autocrine or paracrine loops involving the IGF-R and its
ligand/s may be crucial determinants for the in vivo growth
and tumorigenicity of transformed epithelial cells.
To date, few definitive data have characterized the synthesis and secretion of IGFs from human cancer cell lines. Most studies carried out on the mitogenic and autocrine effects of the IGFs have utilized exogenous addition of IGF-I(4, 8) . For lung cancer cells, this approach was initially supported by work from several groups who reported small (milliunits/milliliter) amounts of ``IGF-I-like immunoreactivity'' in conditioned growth medium from lung cancer cell lines(11, 12) . However, these original studies are now recognized to be confused by IGF-binding proteins (IGFBP), by IGF-II cross-reactivity with the antibodies used for the IGF-I measurements(13) , and have been contradicted by later analysis of a small number of lung cancer cell lines from one of these groups(11) , which suggests low and infrequent expression of IGF-I mRNA and a higher level of IGF-II mRNA expression. Comparing the data in these and other papers, it is apparent that IGF-R is frequently expressed(7, 14) , but the identity of the relevant ligand is still unclear; the IGF-I-like immunoreactivity is not IGF-I (cell lines positive for immunoreactivity are negative for mRNA expression by RT-PCR), and there is post-transcriptional regulation of synthesis and/or secretion of the IGFs (cell lines positive by RT-PCR and Northern analysis are negative for secreted immunoreactivity)(11, 12, 15) .
Elucidation of the identity of the critical IGF-R ligand is necessary for potential early tumor detection and therapeutic interventions in a wide range of human malignancies. In order to determine the identity of the secreted IGF involved in cancer cell autocrine growth, we have carried out an extensive characterization of the expression of IGF mRNA, and the synthesis, processing, and release of IGF peptides by human lung and breast epithelial cancer cell lines. For these studies we used both Northern analysis and reverse transcription of mRNA followed by PCR (RT-PCR) to examine gene expression. We also purified to homogeneity several differentially processed and glycosylated IGF peptides secreted by human cancer cell lines, and we have definitively identified the peptides by amino-terminal Edman sequence analysis.
The
single IGF-I gene comprises five exons, which are alternatively spliced
to generate two mRNA transcripts(18) . The two translation
products, the IGF-IA and IGF-IB prohormones, differ in the region that
encodes the carboxyl-terminal extension peptide (E-peptide), which is
cleaved from pro-IGF-I during biosynthesis(18) . Reactions for
amplification of IGF-I cDNA were primed with an oligonucleotide
complementary to the common mRNA region, which encodes the IGF-I
NH terminus, and antisense primers were complementary to
sequences in the IGF-IA and IGF-IB splice variants. The primers had the
following sequences: IGF-I sense, 5`-GGACCGGAGACGCTCTGCGG-3`; IGF-IA
antisense, 5`-TCTACTTGCGTTCTTCAAAT-3`; IGF-IB antisense,
5`-TTTGCCTCTGCATTCAGCAT-3`. The IGF-II gene encodes a single
prepro-IGF-II, from which the mature 67 amino acid IGF-II is cleaved
during biosynthesis(19) . Primers for the amplification of
IGF-II cDNA sequences were as follows: IGF-II sense,
5`-AGTCGATGCTGGTGCTTCTCA-3`; IGF-II antisense,
5`-GTGGGCGGGGTCTTGGGTGGGTAG-3`.
The integrity of the RNA and the
efficiency of RT-PCR were monitored by amplifying the cDNA with primers
specific for human type I IGF-R, which is ubiquitously expressed by
human lung cancer cell lines. The primers for amplification of IGF-R
were as follows: IGF-R sense, 5`-ATTGAGGAGGTCACAGAGAAC-3`; IGF-R
antisense, 5`-TTCATATCCTGTTTTGGCCTG-3`. PCR reaction mixes consisted of
2 µl of cDNA, 100 ng each of 3` and 5` primer, 2.5 units of Taq polymerase (Ampli-Taq, Applied Biosystems Division, Perkin-Elmer)
in 100 µl of PCR buffer (1.5 mM MgCl, 200
µM dNTPs, 50 mM KCl, 10 mM Tris-HCl, pH
8.3). For amplification of IGF-I sequences, 35 cycles of PCR was
programmed as follows: 94 °C, 15 s/50 °C, 15 s/72 °C, 15 s;
followed by a final 5-min extension at 72 °C. For PCR with IGF-II
and IGF-R primers, the annealing temperature was raised to 60 °C.
Gels were Southern blotted onto 0.2-µm nitrocellulose
(Schleicher & Schull) in 20 SSC, baked at 80 °C for 2
h, and hybridized overnight at 42 °C with 10
cpm/ml
oligonucleotide probe complementary to sequences found between the PCR
primers. The sequence of the antisense oligonucleotide probes were as
follows: IGF-IA probe, 5`GCGCTCGGCACGGACAGAGCG; IGF-IB probe,
5`-TCCAATCTCCCTCCTCTGCT-3`; IGF-II probe, 5`AGGCGCTGGGTGGACTGC-3`;
IGF-R probe, 5`-GTACTCTGTCTCCAGCTCTTC-3`. The probe was end-labeled
with [
-
P]ATP using T4 polynucleotide kinase
(Life Technologies, Inc.) and diluted in hybridization
buffer-N(17) . After hybridization the blots were washed at
room temperature twice in 2
SSC and once in 0.5
SSC.
Autoradiography was performed at -70 °C on Kodak X-AR film
with an intensifying screen. PCR product derived from cell line H1385
was cloned into the TA cloning vector (Invitrogen, San Diego, CA) and
sequenced using the dsCycle sequencing kit (Life Technologies, Inc.)
according to the manufacturer's instructions.
Figure 1: IGF and IGF-R gene expression in lung cancer cell lines as detected by RT-PCR (A) and IGF and IGF-R gene expression in the breast cancer cell lines (B). RT-PCR products were electrophoresed on agarose gels, Southern blotted, and probed with specific oligonucleotide probes complementary to an internal sequence to confirm the identity of the PCR products. The faster migrating band of the doublet visible with the IGF-IB primers is single-stranded PCR product; when excised and reamplified, it yielded a product that migrated with the upper band. The figure shows a subset of the data used to generate Table 1. Some bands not visible in the photograph were seen on longer exposure of the probed Southern blot to film.
Figure 2:
Specificity of anti-IGF antibodies for the
detection of recombinant human IGF-II and IGF-I. Varying amounts of the
rhIGF standards as indicated were electrophoresed on 10-20%
Tricine SDS-PAGE gels and Western blotted. Blots were incubated with
either the anti-IGF mAb or the polyclonal
anti-IGF.
In the initial screening of 500 ml of RCM,
immunoreactivity was detected in medium conditioned by only 3/10 cell
lines: NCI-H820, NCI-H2087, and NCI-H2380. Fig. 3A shows the chromatogram obtained when R
CM from the cell
line NCI-H2087 was fractionated on a C
column. IGF
immunoreactivity (Fig. 3B) was in present as three
bands of 23, 14, and 6-kDa and eluted in fractions 34-36. When
IGF-I and IGF-II standards were chromatographed on the preparative
C
column, they had the same retention time as the
immunoreactivity from R
CM and they co-migrated with M
6000 species on the mini-gels used for the
immunoblotting (data not shown). The relative intensity and
distribution of the three immunoreactive species varied between these
cell lines (Fig. 3C). Electrophoresis under reducing
conditions did not alter the apparent size of the three immunoreactive
species, suggesting that they are monomeric species.
Figure 3:
Western blot analysis of RCM
concentrated by RP-HPLC using anti-IGF mAb. A, chromatograph
of 500 ml of H2087 R
CM separated on C
RP-HPLC; B, Western blot analysis of fractions from chromatography of
H2087 R
CM analyzed with anti-IGF monoclonal antibody; C, IGF immunoreactivity in fractionated R
CM from
lung and breast cancer cell lines (500 ml each). Western blots were
probed with the anti-IGF mAb, and relative amounts of each
immunoreactive species were quantitated using a PhosphorImager.
&cjs2112;, 23-kDa form; &cjs2113;, 14-kDa form;
, 6-kDa
form.
When larger
volumes of RCM (3-7.5 liters) from the cell lines
NCI-H345 and NCI-N417 were concentrated on the C
column,
weak immunoreactive bands corresponding to M
6000
were detected after extended exposure of Western blots (data not
shown). R
CM from the cell lines NCI-H187 (2 liters),
NCI-H1385 (1.5 liters), NCI-H510 (3 liters), MCF-7 (2 liters), and
NCI-H460 (3 liters) tested negative for IGF immunoreactivity.
Figure 4:
Specificity of immunoreactivity detected
by Western blotting. Top panel, H2087 RCM was
concentrated by RP-HPLC as in Fig. 3A, and samples of
the peak immunoreactive fraction were Western blotted under the
conditions shown. Bottom panel, H2087 R
CM with 5
µg of rhIGF-I added (final concentration 1.2 nM) was
concentrated by RP-HPLC as for the top panel. Lane a,
probed with anti-IGF mAb (1 µg/ml); lane b, probed with
anti-IGF mAb (1 µg/ml) + 1 µM rhIGF-I; lane
c, probed with 1/1000 polyclonal
anti-IGF-I
.
Figure 5:
Separation of IGF immunoreactive species
by RP-HPLC. A, phenyl RP-HPLC of pooled IGF-containing
fractions from NCI-H2087 RCM concentrate eluted with a
gradient of acetonitrile in 0.1% HFBA; B, Western blot of
fractions analyzed with the anti-IGF mAb.
Previous in vitro studies have suggested that an
IGF/IGF-R autocrine loop may be operating in many human epithelial
cancer cell lines. The inhibitory effect of the anti-IGF-R antibody,
-IR3, on the growth of lung cancer cell lines has been documented (4, 10) , and studies by others and ourselves have
confirmed the inhibitory effect of this antibody on the clonal growth
of the breast cancer cell line MCF-7 in semi-solid agar in the presence
of serum (8) and the absence of added serum or insulin-like
peptides. (
)Our previous studies have shown that IGF
stimulation of tumor cell growth is mediated via an active
IGF-R(4) . Recent work has confirmed the importance of
IGF/IGF-R interactions in maintenance of the transformed phenotype and
control of apoptosis(1, 2, 3) . The capacity
of the lung and breast cancer cell lines investigated in these studies
to adapt to growth in unsupplemented basal medium with minimal changes
in the pattern of IGF gene expression suggests that IGF/IGF-R autocrine
loops may be constitutive elements which influence the growth
characteristics of these tumors. As IGF-R is expressed by most actively
growing cells, elucidation of the identity of the synthesized and
secreted IGF ligand is crucial to our understanding of epithelial tumor
biology.
The two mature 7.5-kDa IGF peptides exhibit close to 70% amino acid identity with each other and are 50% homologous with pro-insulin(22) . Studies have implicated both IGFs in the growth and differentiation of normal epithelia. In the developing fetal lung, IGF-II appears to play a critical role, since mice lacking a functional IGF-II gene died at birth due to a failure of lung inflation(5) . In situ hybridization studies have localized IGF-I expression to the mesenchymal compartment of adult and developing lung, suggesting that IGF-I may influence airway development via paracrine modes of action (23) . Corroborating evidence has demonstrated the secretion of IGF-I by primary cultures of human lung fibroblasts(24) . These findings suggest that the pattern of IGF expression in lung neoplasms may parallel that of the breast epithelium, where IGF-I is principally expressed by stromal tissue while IGF-II is expressed by epithelial tumor cells (25) . Elucidation of the role of IGFs in autocrine growth is complicated by the synthesis of IGFBP which are expressed by many cancer cell lines, usually in large excess over the IGFs(14, 15, 26) . The IGFBP therefore confound interpretation of early studies, which aimed to use anti-IGF antibodies to investigate the effect of IGF neutralization on cell growth. Using purification and assay methodologies that obviate interference by IGFBP, we have demonstrated conclusively the secretion by human lung and breast cancer cell lines of ligand/s that can bind and activate the IGF-R.
Purification, SDS-PAGE, and
NH-terminal Edman sequencing demonstrated IGF-II peptide
secretion in the breast adenocarcinoma cell line (H2380), and two of
the lung cancer cell lines investigated: an adenocarcinoma (NCI-H2087)
and a bronchoalveolar carcinoma (NCI-H820). IGF-I peptide sequence was
obtained from an M
6000 species isolated from
medium conditioned by a variant SCLC cell line, NCI-N417. IGF-II was
secreted as multiple molecular weight forms, as has been described for
IGF-II present in normal adult serum and serum from hypoglycemic
patients bearing mesenchymal tumors(27) . The
post-translational processing of the primary IGF-II prepropeptide is
complex and can yield multiple molecular weight forms of IGF-II, the
nature of which is determined by differential post-translational
cleavage and glycosylation events(19, 28) . The 20-kDa
preproprotein is sequentially processed by cleavage at a single lysine
residue at position 21 of the prohormone to yield a 10.5-kDa peptide
(pro-IGF-II E21), which has been isolated from normal human
serum(29) , and then by removal of the E-peptide to yield
mature IGF-II. A 15-kDa form of pro-IGF-II E21 has also been isolated,
which includes O-linked sialic acid residues attached to
Thr
of the E-peptide(28) .
Studies are
continuing to characterize fully the exact nature of the 14- and 23-kDa
IGF-II species. The 6-kDa form identified here, which co-migrated on
SDS-PAGE with the 7.5-kDa rhIGF-I/II standards, was identified as fully
processed mature IGF-II. The primary IGF-II translation product has
been calculated to be a 20-kDa polypeptide (30) . The 23-kDa
form of IGF-II isolated here could represent the unprocessed precursor,
which may be running with an artifactually high molecular mass on
SDS-PAGE gels, or could be due to glycosylation at Thr of
the E-peptide region of the intact prepropeptide. Preliminary data
indicates that the 14-kDa species, isolated from the H2087
adenocarcinoma, can be converted to a species migrating at 10 kDa on
SDS-PAGE after incubation with O-glycanase and neuraminidase
(data not shown). It is therefore equivalent to the 15-kDa IGF-II
characterized by Hudgins et al.(28) . This
glycosylated and sialated 15-kDa IGF-II has higher potency for growth
stimulation and receptor activation than the 7.5-kDa peptide and
altered interactions with IGFBP(27, 31) . At least two
of the three forms of IGF-II isolated from these lung and breast tumor
cell lines are therefore capable of acting as autocrine growth factors.
From the incidence of IGF gene expression shown in Table 1, it
would appear that IGF autocrine loops are operating in the majority of
the cell lines, i.e. IGF-R was co-expressed with one or both
of the IGF peptide genes. IGF-II peptide secretion was detected in 3 of
the 10 cancer cell lines intensively investigated. These three were
cell lines with IGF-II mRNA levels detectable by the less sensitive
Northern blot procedure. All of the cell lines investigated, with the
exception of the SCLC cell line NCI-H187 and the breast line MCF-7,
expressed one or both of the IGF-I transcripts. However, we were only
able to definitively identify IGF-I peptide secretion by the SCLC line
NCI-N417d. In the cell lines from which IGFs were not isolated, we
calculate (based upon the sensitivity of the antibodies for the
detection of the IGFs) that IGF peptides may only accumulate in CM at
subpicomolar concentrations. As IGF-R inhibition studies with mAb
-IR3 demonstrate active IGF/IGF-R autocrine loops in many of the
cell lines studied here(4) , mechanisms could be operating by
which a low level of synthesized IGF is preferentially delivered to the
receptor and rapidly turned over, resulting in accumulation of
synthesized IGF at levels too low for measurement using present
techniques. Such mechanisms may be potentiated by the IGFBP, which we
found to be secreted by human lung cancer cell lines in large excess of
the endogenously secreted IGFs. Membrane-bound forms of IGFBP, which
have been identified in lung cancer cell lines(14) , may play a
role in the delivery of IGFs to the receptor.
Addendum-Since this work was submitted for publication, a report on a single prostate cancer cell line has suggested that IGF-II/IGF-R is involved with autocrine growth regulation in that epithelial system(32) , and a recent study demonstrated that an IGF-II/IGF-R autocrine loop mediates epidermal growth factor-induced proliferation in a cervical cancer epithelial cell line(9) . Those findings, as well as the conclusive identification of multiple active forms of IGF-II synthesized and secreted by both lung and breast epithelial cancer cell lines reported here, point to the conservation of the IGF-II/IGF-R autocrine pathway as a central mechanism in the process of epithelial carcinogenesis.