characterization of an antibody that can detect an activated igf-i receptor in human cancers
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Experimental Cell Research 251, 22–32 (1999)Article ID excr.1999.4562, available online at http://www.idealibrary.com on
Characterization of an Antibody That Can Detect an Activated IGF-IReceptor in Human Cancers
Michele Rubini,* Consuelo D’Ambrosio,† Sabrina Carturan,* Gladys Yumet,‡ Edison Catalano,§ Simei Shan,‡Ziwei Huang,‡ Mario Criscuolo,† Micol Pifferi,† and Renato Baserga‡,1
*University of Ferrara, Via L. Borsari 46, 44100 Ferrara, Italy; †University of Modena, Via del Pozzo 71, 41100 Modena, Italy;‡Kimmel Cancer Center, Thomas Jefferson University, 233 S. 10th Street, 624 B.L.S.B., Philadelphia, Pennsylvania 19107;
and §Cooper Hospital University Medical Center, One Cooper Plaza, Camden, New Jersey 08103
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The type 1 insulin-like growth factor receptor (IGF-R) plays an important role in malignant transforma-ion and in apoptosis. Its role in human cancer hasow been firmly established. IGF-IR signaling occursnly when the receptor is activated by its ligands,hich induce autophosphorylation of the receptor at
everal tyrosine residues. Although the IGF-IR (phos-horylated or not) can be detected in human cancersith conventional antibodies, it would be desirable tobtain antibodies that can detect the IGF-IR onlyhen activated by its ligands. We describe and char-cterize in this paper such an antibody and show thatt can be used in sections of human cancers to detectn autophosphorylated IGF-IR. This antibody will beseful in detecting autocrine or paracrine influencesn normal and tumor cells and could eventually belso useful in diagnostic and prognostic studies of hu-an primary and metastatic cancer. © 1999 Academic Press
INTRODUCTION
The insulin-like growth factor receptor (IGF-IR) isnown to play an important role in the transformationnd survival of cells, both in vivo and in vitro [1].ver-expression and/or constitutive activation of
GF-IR in a variety of cell types leads to ligand-depen-ent growth in serum-free medium and to the estab-ishment of a transformed phenotype; i.e., ability toorm colonies in soft agar and/or to produce tumors inice [2, 3]. When the function of the IGF-IR is de-
reased or otherwise impaired by antisense strategiesr by dominant negative mutants, or by triple-helixormation, there is a dramatic inhibition of tumorrowth [4–11] and metastases [12–14] in experimentalnimals. The inhibition of tumor growth is largely dueo the fact that the IGF-IR protects cells from a varietyf apoptotic injuries, and, as a consequence, when the
1 To whom correspondence and reprint requests should be ad-
hressed. Fax: (215) 923-0249. E-mail: [email protected].22014-4827/99 $30.00opyright © 1999 by Academic Pressll rights of reproduction in any form reserved.
eceptor is impaired, tumor cells are more susceptibleo cell death. Thus, addition of IGF-I and/or overex-ression of the IGF-IR protect hemopoietic cells frompoptosis caused by interleukin-3 (IL-3) withdrawal15–19]. IGF-I also protects cells overexpressing c-myc,hich undergo apoptosis in serum-free medium and in
he presence of other growth factors [20]. Finally, anctivated IGF-IR protects human cancer cells from celleath induced by diverse anti-cancer drugs [21], etopo-ide [22], tumor necrosis factor a [23], transformingrowth factor b [24], p53 [25], ionizing and non-ioniz-ng radiation [26–28], okadaic acid [29], and serumeprivation [30].The IGF-IR is ubiquitous, and the only two cell types
hat, so far, are known to be devoid of IGF-IR areepatocytes and B lymphocytes [1]. Although there areubstantial amounts of IGF-I and IGF-II in the plasma31], it is now generally accepted that these ligands are
ostly bound to a number of IGF-binding proteins [32]nd that the activation of the IGF-IR is usually due toutocrine or paracrine influences [1]. The IGF-IR cane detected in tissue sections by the use of appropriatentibodies, and in fact, it has already been used tohow that it is a significant prognostic factor in humanreast cancer [27]. Other reports have confirmed themportance of the IGF system in human cancer [33,4], especially in breast cancer [35–38], and prostateancer [39]. Because of the importance of the IGF sys-em in cancer, it would be desirable to develop antibod-es against the IGF-IR that could become useful tools inhe diagnosis and/or prognosis of malignant diseases.
The antibodies presently available detect the IGF-IRn cells regardless of its state of activation. In immu-oblots, the activated receptor can be visualized, afterpecific immunoprecipitation, with an anti-phosphoty-osine antibody, but this procedure is not feasible inissue sections. We therefore decided to develop anntibody that could specifically detect a tyrosyl phos-horylated IGF-IR, i.e., an IGF-IR activated by its li-ands, in tissue sections from normal or cancerous
uman tissues. There are certain limitations to this
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23IGF-I RECEPTOR AND CANCER
ndeavor: the IGF-IR and the insulin receptor (IR)ave extensive homologies in the tyrosine-rich regionsf the kinase domain and the juxtamembrane domain40]. In the kinase domain, the homology is close to00%, making it very difficult to obtain antibodies thatould distinguish between the two receptors. However,n the C-terminus, homology between the two receptorsalls to 44%, and we therefore selected this region toroduce an antibody to the tyrosyl phosphorylated C-erminus of the IGF-IR. We describe and characterizeere this antibody, and we demonstrate its ability toetect an activated IGF-IR in tissue sections of humanancers.
MATERIALS AND METHODS
Peptide synthesis. As described previously [41, 42], the peptidesere prepared by solid-phase synthesis using Fmoc strategy on a30A peptide synthesizer (Applied Biosystems, Foster City, CA) and
9050 Pepsynthesizer Plus (Perseptive Biosystems, Cambridge,A). A 4-fold excess of Na-Fmoc amino acid, O-benzotriazol-1-yl-,N,N9,N9-tetramethyluronium hexafluorophosphate, and 1-hy-roxybenzotriazole and a 10-fold excess of diisopropylethylamineere used in every coupling reaction step. Removal of the NH2-
erminal Fmoc group was accomplished by 20% piperidine in N,N-imethylformamide. The cleavage of peptides from the resin wasarried out with reagent K [43] for 2 h at room temperature withentle stirring. Crude peptides were precipitated in ice-cold methyl--butyl ether, centrifuged, and lyophilized. Crude peptides were thenurified by preparative reverse-phase HPLC using a Dynamax-300Å18 column (25 cm 3 21.4 mm, inner diameter) with a flow rate of 9l/min and two solvent systems of 0.1% trifluoroacetic acid/H2O and
.1% trifluoroacetic acid/acetonitrile. The fractions containing theppropriate peptide were pooled together and lyophilized. The purityf the final products was assessed by analytical reverse-phase HPLC,apillary electrophoresis, and matrix-assisted laser desorption/ion-zation time-of-flight mass spectrometry.
Pep922 was a 19-amino-acid peptide corresponding to the C-ter-inus residues 1319–1337 of the human IGF-IR (numbering ofllrich et al., ref. 40). Pep1046 was a 28-amino-acid peptide corre-
ponding to residues 1310-1337 of the human IGF-IR, including ahosphotyrosyl residue at position 1316. Its sequence is NH2, FDER-PpYAHMNGGRKNERALPLPQSSTCCOOH.Immunization protocol and antibodies. Peptides were dissolved
n 0.1 M sodium phosphate buffer (pH 7.0) and coupled to activatedeyhole limped hemocyanin (Pierce) by glutaradehyde. Immunogensere diluted 1:1 with complete (first shot) or incomplete (subsequent
hots) Freund’s adjuvant and injected intradermally in rabbits at-week intervals. The anti-peptide serum antibody level was moni-ored by ELISA. To get rid of unwanted anti-C terminus antibodies,nti-pep1046 serum was cleared by reverse immunoaffinity purifi-ation using pep922-bound Sepharose. The antibody againstep1046 is hereafter referred to as anti-pY1316.For detection of the IGF-IR b-subunit, an anti-C-terminus IGF-IR
ntibody (Santa Cruz) was used. We also used an antibody obtainedsing anti-pep922 antiserum; it behaved exactly as the commercialntibody against the C-terminus. For detection of the overall tyrosylhosphorylation of immunoprecipitated receptors, the PY20 anti-ody (Transduction Labs.) was used. Other antibodies are describedn the section on immunoblots.
Cell cultures. Cells were maintained in Dulbecco’s modified Ea-le’s medium (DMEM) supplemented with 10% bovine calf serum,lutamine (20 mg/ml) MEM, penicillin (10 U/ml), and streptomycin
0.1 mg/ml). Incubations were at 37°C in a CO2 incubator under otandard conditions. To make cells quiescent, they were cultured fordays in serum-free medium (DMEM supplemented with 50 mg/ml
ransferrin and 0.1% bovine serum albumin).Immunoprecipitation and immunoblotting. To induce receptor
hosphorylation, quiescent cells were stimulated for 5 min withGF-I (3.3 nM) or with insulin (6.5 nM). Culture dishes were thenlaced on ice and cells were rinsed three times in cold Hanks’. Cellsere lysed in lysis buffer [50 mM Hepes (pH 7.5), 150 mM NaCl, 1.5M MgCl2, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 100 mMaF, 0.2 mM Na3VO4, 10 mM Na4P2O7, 1 mM phenylmethylsulpho-yl fluoride, 10 mg/ml aprotinin] for 4 min at 4°C. Lysates wereollected and cleared from nuclei by centrifugation.Lysates (150 mg of protein) were diluted with HNTG buffer [20 mMepes (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, 10% glycerol, 100M NaF, 0.2 mM Na3VO4, 5 mM phenylmethysulphonyl fluoride, 10g/ml aprotinin] containing 15 ml of protein A–agarose (Calbiochem)nd 10 ml of anti-IGF-IR (Ab-1) monoclonal antibody (Oncogenecience) or 10 ml of anti-insulin receptor (Ab-3) monoclonal antibody
Oncogene Science). Antibody–antigen complexes were allowed toorm for 4 h at 4°C and then collected by centrifugation at 4°C for 4
in. Immunoprecipated complexes were washed three times inNTG and resuspended in 20 ml of Laemmli buffer (20% glycerol, 3%
odium dodecyl sulfate, 3% b-mercaptoethanol, 10 mM EDTA, 0.05%romophenol blue). Samples were boiled for 4 min and proteins wereeparated on 4–20% polyacrylamide gradient gels (Bio-Rad) by so-ium dodecyl sulfate–polyacrylamide gel electrophoresis. Proteinsere electroblotted onto nitrocellulose filters. Membranes werelocked with 5% bovine serum albumin or 5% nonfat milk in TBST10 mM Tris (pH 8.0), 150 mM NaCl, 0.1% Tween 20] overnight at°C, probed with 10 ml of the indicated antibodies in 20 ml TBSTolution for 1 h. When secondary antibody interaction was needed,fter five washes in TBST, filters were probed with 2 ml of anti-rabbitgG-HRP antibody (Oncogene Science) in 20 ml TBST solution forh. After washing five times in TBST, blots were developed with the
nhanced chemiluminescence detection system (Amersham) accord-ng to the manufacturer’s instructions. Immunoblot reprobing wasone after filters were incubated for 30 min in stripping buffer [62.5M Tris (pH 6.8), 2% SDS, 100 mM b-mercaptoethanol] at 50°C and
eblocking overnight at 4°C in 5% nonfat milk–TBST.Peptide competition test. Anti-pep1046 (anti-pY1316) antibodyas challenged with pep922, or the commercial antibody to the-terminus (Santa Cruz), or pep1046 itself. Five microliters of anti-ep1046 antibody was incubated with 8 ml of 0.1 M Na-phosphateuffer (PB), pH 7.0, and 10 ml of 10 mg/ml pep922, commercialntibody, pep1046, or just PB. Incubation was for 30 min at 37°C.ach reaction was used to probe immunoprecipitated IGF-IR immu-oblots.Staining of tissue sections. Tissue sections (5 mm thick) were
btained from paraffin-embedded tumor specimens. After standardeparaffinization, the sections were probed either with an anti-Y1316 antibody or with an antibody to the C-terminus of the IGF-IRanti-pep922 antibody). After incubation with goat anti-rabbit IgGiotinylated antibody and peroxidase-conjugated streptavidin, themmune complex was visualized with the chromogenic substrateAB (diaminobenzidine tetrahydrochloride). Several controls were
ncluded (preimmune serum, peptide competition), and consecutiveections were examined.
RESULTS
For clarity, we designate as antibody anti-pY1316,he antibody developed against the 1046 peptide de-cribed under Materials and Methods, which containsphosphorylated tyrosine 1316. We shall refer to the
ther antibodies that recognize the C-terminus of the
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24 RUBINI ET AL.
GF-IR (regardless of the status of its phosphorylation)s anti-C-terminus antibodies.
he Anti-pY1316 Antibody Recognizes an Epitopein the C-terminus of the IGF-IR
The first test was to determine whether this anti-Y1316 antibody recognized the intended epitope inhe C-terminus of the IGF-IR. For this purpose, wesed three cell lines, all derived from R2 cells [44, 45],hich are 3T3-like cells originating from mouse em-ryos with a targeted disruption of the IGF-IR genes46, 47]. The three cell lines were the parental R2 cells,c-4 cells, which are R2 cells stably transfected with anGF-IR truncated at residue 1229 [48], and R1 cells,hich are R2 cells stably transfected with a wild-typeuman IGF-IR cDNA [44]. Lysates were prepared fromhese cells stimulated or not by IGF-I. In Fig. 1, allysates were first immunoprecipitated with an anti-ody to the a subunit of the IGF-IR (see Materials andethods), which precipitates both subunits. In Fig. 1,
ottom, the blot was stained with an antibody to the-terminus of the IGF-IR (anti-pep922 antibody): asxpected, only lysates from R1 cells gave a signal, sincehis antibody will not recognize R2 cells (no receptor) orc-4 cells (missing the C-terminus). In Fig. 1, top, thelot was stained with the anti-pY1316 antibody. Again,nly R1 cells give a positive signal, since the Y1316 isissing from the receptor of Tc-4 cells (and obviously
rom R2 cells). The antibody against the C-terminusives bands of similar intensity, regardless of the ac-ivity status of the receptor, while the anti-pY1316
FIG. 1. Specificity of the anti-py1316 antibody. In all instances,he IGF-I receptor was immunoprecipitated from lysates with anntibody to the a subunit of the IGF-IR (Oncogene Science). Lysatesere prepared from three types of cells: R2 (no IGF-IR); Tc-4 (IGF-IR
runcated at residue 1229); and R1 (full-length IGF-IR). The lysatesere prepared from cells that were unstimulated (2) or were stim-lated for 5 min with IGF-I (3.3 nM). (Top) Blot with an antibody toY1316. (Bottom) Blot with an antibody to the C-terminus of theGF-IR.
ntibody gives a much stronger signal when the lysates e
re prepared from IGF-I-stimulated cells. The faintand in unstimulated cells is due to the fact that R1
ells secrete a small amount of IGF-I [44], which pro-uces a background activation of the receptor (see be-ow). Therefore, the anti-pY1316 antibody can detectn autophosphorylated wild-type receptor, but does notetect an IGF-IR lacking the C-terminus (the last 108mino acids).
pecificity of the Anti-pY1316 Antibody
The anti-pY1316 antibody was then tested on aanel of R2-derived cells expressing a number of mu-ants of the IGF-IR. These mutant receptors and theell lines derived from them have been described inetail in previous papers [49–53] and are summarizedn Table 1. The cells were left unstimulated (see Ma-erials and Methods) or were stimulated with IGF-I3.3 nM) for 10 min. Figure 2 summarizes the resultsbtained by immunoprecipitating the lysates with anntibody to the a subunit of the IGF-IR (which immu-oprecipitates both subunits) and blotting with thenti-pY1316 antibody (top), or an anti-C-terminus an-ibody (middle), or an anti-phosphotyrosine antibodybottom). R2 cells are negative with all three antibod-es, as expected. An antibody to the C-terminus of theGF-IR (middle) detects the IGF-IR in all cell lysates,xcept those of R2 cells and the cells expressing the1245 mutant. The R2/d1245 cells [53] have a receptorruncated at residue 1245, and the epitopes for the twontibodies used to detect the b subunit are both down-tream from residue 1245. This mutant receptor,
TABLE 1
Cell lines Mutant human IGF-IR
Densitometricratios
pY pY1316
2 Null-/-IGF-IR 0 02/950F Y950F 11.9 4.12/KA K1003A 0 02/YF1 Y1131F, Y1135F, Y1136F 6.1 1.92/1245D Truncation at 1245 6.0 02/Y1250F Y1250F 4.9 2.22/Y1251F Y1251F 1.5 1.52/Y1250/1F Y1250, Y1251F2/S1280-3A S1280-83 to A 29.6 12.52/1316F Y1316F 4.0 01 wt 68.7 30.1-IR9 Null-/-IGF-IR, overexpressing
human insulin receptor
Note. The development of the mutant receptors and the character-zation of the cell lines have been described in previous papers49–54]. The densitometric ratios give the fold increase in densito-etric intensities of the autophosphorylated IGF-IR bands over thenstimulated cells. The higher the increase in ratio, the higher is the
xtent of autophosphorylation of the activated receptor.
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25IGF-I RECEPTOR AND CANCER
hough, is visible when the membranes are blottedith an anti-phosphotyrosine antibody. The band is, asxpected, lower than the bands of the other full-lengthsubunits.When the cells are stimulated with IGF-I, the anti-
hosphotyrosine antibody detects the receptor in allell lines, except R2 cells and R2/KA cells. R2/KA cells54] have an IGF-IR with a mutation at lysine 1003,he ATP-binding site. This receptor does not autophos-horylate after addition of IGF-I and is essentially annactive receptor. With the anti-pY1316 antibody, noignal is detectable in R2 cells, in R2/KA cells, d1245ells, and the Y1316F cells. The crucial cell line is the
2/Y1316F cells [53], which have a receptor with autation at Y1316 from tyrosine to phenylalanine and
re therefore missing Y1316. No signal is detectableith the anti-pY1316 antibody, while the other twontibodies (phosphotyrosine and b subunit) give a goodignal.Therefore, from Fig. 2, we can conclude that one
eeds a phosphorylated Y1316 for the anti-pY1316 an-ibody to be able to recognize the IGF-IR. This is con-rmed by the finding that in all other mutants, thenti-pY1316 antibody recognized an autophosphory-ated receptor. These include, besides R1 cells, cellsxpressing the following mutant receptors: a Y950Futant [49]; a mutant at the three tyrosines of the
inase domain [51], in which autophosphorylation isecreased but not abolished; mutant receptors of the-terminus, like the Y1250F, the Y1251F, or both1250 and Y1251 [50]; and a mutant at serines 1280–283 [52]. In some cell lines, the anti-pY1316 antibodyives a faint band even in unstimulated cells, which isue, as explained above, to residual IGF-I or IGF-IIecreted by the cell lines in question [44, 54]. Indeed, in
FIG. 2. Detection of mutant receptors by the anti-pY1316 annstimulated (2) or stimulated with IGF-I (1). The lysates were immere stained with either the anti-pY1316 antibody (first row), ornti-phosphotyrosine antibody (third row). The antibodies are descr
ll those cases, the anti-phosphotyrosine antibody also a
ave a positive signal, indicating that the receptor hadeen activated.
ensitometric Analysis
The intensity of the bands obtained with the anti-hosphotyrosine antibody, with the anti-pY1316 anti-ody, and with the antibody to the C-terminus of theGF-IR were quantitated by densitometry. The ratio ofensitometric values between unstimulated cells andGF-I-stimulated cells (after correction for receptorevel) is given in Table 1, with the list of mutant recep-ors. The ratios vary, depending in part on how quies-ent the cells were, but Table 1 clearly shows that: (1)n R2 and R2/KA cells, the ratios do not changehether the cells are or are not stimulated with IGF-I;
2) the d1245 receptor and the Y1316F receptor show aharp increase in autophosphorylation, when mea-ured with the anti-phosphotyrosine antibody, but noncrease when measured with the anti-pY1316 anti-ody; and (3) with the other mutant receptors, auto-hosphorylation increases are detectable with eitherntibody. It is true that the increases are larger withhe anti-phosphotyrosine antibody, but this can be ex-lained by the fact that the anti-phosphotyrosine anti-ody recognizes all the phosphorylated tyrosines of theeceptor, while the anti-pY1316 antibody recognizesnly one tyrosine. It seems therefore that the anti-Y1316 antibody specifically recognizes an autophos-horylated IGF-IR with a phosphorylated Y1316.
he Anti-pY1316 Antibody Does Not Cross-reactwith the Insulin Receptor
In the Introduction, we have given the rationale forhoosing the peptide used to generate the anti-pY1316
dy. For every cell line, lysates were prepared from cells eitherprecipitated with an antibody anti-IGF-IR (a subunit), and the blots
antibody to the C-terminus of the IGF-IR (second row), or anunder Materials and Methods, and the cell lines in Table 1.
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ntibody. One of the main criteria was that the se-
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26 RUBINI ET AL.
uence chosen should be sufficiently different from anyequence in the insulin receptor [40]. This is impor-ant, especially in view of the report that some anti-eptide antibodies can recognize similar receptors [55].igure 3 shows that the anti-pY1316 antibody canistinguish between the activated IGF-IR and the ac-ivated insulin receptor (IR). The cells used were R2
ells expressing a very high level of insulin receptors.his cell line has been previously described [50, 56].he cells were stimulated or not with insulin, the ly-ates were immunoprecipitated with an antibody to theR, and the blots stained with either an anti-phospho-yrosine antibody (Fig. 3, bottom) or with the anti-Y1316 antibody (top). It is clear that the anti-pY1316ntibody cannot recognize the autophosphorylated IR,ven in cells that express roughly 5 3 105 IR/cell [56],very large number of IR, about 5 log above the phys-
ological levels of IR in mouse embryo fibroblasts. For aomparison, we show again lysates from R1 cells,here the IGF-IR is detected by both the anti-phospho-
yrosine antibody and the anti-pY1316 antibody. Thepper band (with either the IR or the IGF-IR) is knowno be the proreceptor form, which is frequently visiblehen cells are overexpressing the receptors.
eptide Competition
To confirm that the anti-pY1316 antibody specifi-ally recognizes the sequence used for immunization,e tried competition experiments. In Fig. 4A, the ly-
FIG. 3. The anti-pY1316 antibody does not recognize the insulineceptor. Preparation of lysates, antibodies, and blotting are de-cribed under Materials and Methods. The cell line examined here isR9 cells (no IGF-IR, about 5 3 103 insulin receptors/cell). Lysatesrom cells stimulated (1) or not (2) with insulin were immunopre-ipitated with an antibody anti-insulin receptor. (Top) Blot stainedith an antibody anti-pY1316. (Bottom) Blot stained with an anti-hosphotyrosine antibody. For control, we used R1 cells (see above),here the lysates were immunoprecipitated with an antibody to the
GF-IR a subunit.
ates were immunoprecipitated with anti-a subunit a
ntibody to the IGF-IR and then immunoblotted withhe anti-pY1316 antibody. A band corresponding to theosition of the IGF-IR is clearly evident in lysates of R1
ells stimulated with IGF-I (lane A1). When the im-unoblot is pretreated with the same peptide used for
mmunization, the band is markedly decreased in in-ensity (lane B1). For control, we have used the sameeptide sequence, but without a phosphorylated1316. The band is now again visible in lysates of
GF-I-stimulated R1 cells (lane C1).
he Anti-pY1316 Antibody Can Immunoprecipitatethe IGF-IR
We next tested the ability of this antibody to immu-oprecipitate the IGF-IR. The results are shown in Fig.B, where lysates from R1 cells were immunoprecipi-ated with the anti-pY1316 antibody, before or aftertimulation with IGF-I. The blots were then stainedith an anti-phosphotyrosine antibody. A band corre-
ponding to the b subunit of the IGF-IR is visible aftertimulation with IGF-I (lane 2). The highest band ishe proreceptor. We have not identified the intermedi-te band, whose phosphorylation increases after IGF-I.
etection of the IGF-I Receptor in Human Cancers
We tested our anti-pY1316 antibody on tissue sec-ions of human cancers. The sections were preparednd stained as described under Materials and Meth-
FIG. 4. Further characterization of the anti-pY1316 antibody.A) Peptide competition. IGF-IR immunoblots from IGF-I-stimulated1) or unstimulated R1 cells. The receptor was immunoprecipitatedith an antibody anti-a subunit and immunoblotted with the anti-Y1316 antibody. (A) No competition. (B) Competition with the phos-horylated peptide pep1046 used for immunization. (C) Competitionith control peptide, pep922 (see Materials and Methods). (B) Im-unoprecipitation with the anti-pY1316 antibody. The anti-pY1316
ntibody was used to precipitate the receptor from lysates of IGF-I-timulated (1) or unstimulated (2) R1 cells. The immunoprecipi-ated proteins were immunodetected using an anti-phosphotyrosine
ntibody.
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27IGF-I RECEPTOR AND CANCER
ds. We examined sections of both breast cancers andolon cancers, but, for simplicity (see also below), weill limit ourselves to the documentation of breast
ancers. We ran numerous controls, including the usef a preimmune serum (Fig. 5A), or peptide competitionnot shown, since the specificity of the antibody to theeptide used for immunization has already been docu-ented with a more sensitive method in Fig. 4). Also,e examined consecutive sections, stained with eitherntibody to the C-terminus or an antibody anti-Y1316. For obvious reasons, we are presenting herenly selected pictures, and the selection is admittedlyased on the best and most convincing pictures. FigureB shows a photograph of a human mammary cancer,tained with the anti-pY1316 antibody. Many intra-uctal cancer cells are strongly positive, while the sur-ounding connective tissue is essentially negative. An-ther section of human breast cancer stained with thenti-pY1316 antibody is shown in Fig. 6A. The pictureshown were selected as the best illustrations of thebility of the anti-pY1316 antibody to stain cancerells. In other sections of human cancers, positive can-er cells alternated with cancer cells that were nega-ive or weakly positive. The same can be said of thentibody against the C-terminus of the IGF-IR, ofhich we give an illustration in Fig. 6B.
DISCUSSION
We have developed an antibody, anti-pY1316, thatpecifically recognizes an autophosphorylated IGF-IR.he evidence for this statement can be summarized as
ollows: (1) the antibody recognizes the IGF-IR onlyhen the receptor is phosphorylated, while the corre-
ponding antibody to the C-terminus (nonphosphory-ated) recognizes the IGF-IR regardless of its phos-horylation status; (2) using a panel of several mutantGF-IR, the anti-pY1316 antibody visualizes theGF-IR only when the Y1316 residue is present andhosphorylated; and (3) the anti-pY1316 antibody failso recognize the IR, even after appropriate stimulation.he IR is the only tyrosine kinase receptor that hasome homology to the IGF-IR; the fact that it cannot beetected by the anti-pY1316 antibody even when it isrossly overexpressed rules out any cross-reaction inells with a physiological number of IR. There is an-ther receptor that has substantial homologies withhe IGF-IR (and the IR): the insulin receptor-relatedeceptor (IRRR). However, the IRRR is missing the-termini of the IGF-IR and IR [57]. To be precise, the
RRR terminates at a residue corresponding to residue290 of the IGF-IR. As stated under Materials andethods, our peptide begins at residue 1319 of the
GF-IR.As explained in the Introduction, there are certain
onstraints in developing an antibody specific for an e
utophosphorylated IGF-IR. In the first place, ofourse, one needs a tyrosine (Y) residue that is phos-horylated in the peptide used for immunization. Itould have been better if we could have used the
yrosine kinase domain, which has three Y residues inlose proximity, all of which are autophosphorylated byGF-I. However, the homology with the IR, in this area,s 98%, which makes it very difficult to obtain an an-ibody that will not cross-react with the IR. The Yesidues at 1250/1251 of the IGF-IR could also behought desirable, but the area around 1250/1251 wasound on examination of the sequence to be, at leastheoretically, a poorly antigenic sequence. For theseeasons, we eventually selected a peptide sequence inhe C-terminus of the IGF-IR that has a phosphory-ated tyrosine and little homology to the correspondingequence of the IR.We have shown that the antibody we call anti-
Y1316 is due to immunization with the peptide weynthesized for the purpose. Competition with the orig-nal peptide almost abrogated the immunodetection ofhe autophosphorylated receptor. The properties of thenti-pY1316 antibody are clear. It will detect theGF-IR only when Y1316 is present and phosphory-ated. A mutant receptor in which Y1316 has been
utated is not recognized by the antibody. The samean be said of the d1245 receptor, in which the IGF-IRs truncated at residue 1245. The antibody will notecognize a receptor that cannot be autophosphory-ated, like the KA receptor, in which the IGF-IR has a
utation at lysine 1003, the ATP-binding site [54]. Theutant receptor with mutations at 1131, 1135, and
136 (the tyrosine kinase domain) is known to have aecreased, but not abolished, autophosphorylation51], and this is confirmed in the present experiments.
2 cells are negative for the anti-pY1316 antibody asell as any other antibody raised against the IGF-IR.inally, the anti-pY1316 antibody, besides being useful
n Western blots, is also capable of immunoprecipitat-ng the IGF-IR.
Although the anti-pY1316 antibody will be usefulnder experimental conditions in detecting an auto-hosphorylated antibody directly in Western blots, theeason we developed it is for use on tissue sections ofuman cancers. The IGF-IR is present in many humanancers [33, 34] and sometimes it is overexpressed.owever, the IGF-IR, to transmit its mitogenic andnti-apoptotic signals, must be activated by its ligands.he concentrations of these ligands in the immediateroximity of tumor cells undoubtedly varies, and oneould like to know how many of the cancer cells, at aiven time, actually have an activated (autophosphor-lated) IGF-IR. Our experiments confirm that an anti-ody to the C-terminus of the IGF-IR can easily detecthe receptor in sections of human cancers (we have
lected, in this paper, to show sections of breast cancer,
pc
28 RUBINI ET AL.
FIG. 5. Staining of human breast cancer sections with the anti-pY1316 antibody. (A) A section from a human breast cancer treated withreimmune serum as a negative control. (B) A section from a human breast cancer stained with an anti-pY1316 antibody. The intraductal
ancer cells are intensely positive, while the interstitial tissue is essentially negative.
Aptc
29IGF-I RECEPTOR AND CANCER
FIG. 6. Staining of tissue sections with antibodies against the IGF-I receptor. The antibody used in A was the anti-pY1316 antibody.gain, many intraductal cancer cells are intensely positive, although groups of tumor cells can also be seen that are negative or weaklyositive. The antibody used for the section in B was instead the one obtained with peptide 922 (see Materials and Methods) that recognizeshe C-terminus of the IGF-I receptor, regardless of its phosphorylation status. In both cases, the sections were from cases of human breast
ancer.
bBaTptnpahsaTpbaoewmgdtTactmtastet
tsspdopdiesrpt
aIhspm
atso
t
1
1
1
30 RUBINI ET AL.
ut we could also detect it in metastatic colon cancer).ut we show now that the anti-pY1316 antibody canlso detect the receptor in human breast cancer cells.he next step is to investigate whether the anti-Y1316 antibody can be used for diagnostic or prognos-ic studies, for instance to determine the aggressive-ess of a tumor, or to determine differences betweenrimary tumors and metastases. Prospective studieslong this line have been initiated. Several reportsave emphasized a role of IGF-IR levels in the progno-is of cancer, especially breast cancer [27, 35, 37], andt least two reports have indicated that treatment withamoxifen causes a significant decrease in IGF-Ilasma levels [58, 59]. The anti-pY1316 antibody coulde very useful in following the development of diseasend the efficacy of therapeutic treatment. However,ne should be very careful in studies that have to bextended to a variety of situations. For instance, itould be a mistake to look only at cancers and theiretastases. An activated IGF-IR simply means that
rowth factors (in our case, IGF-I or IGF-II) are pro-uced in relative abundance in the immediate vicini-ies of the positive cells or by the cells themselves.hese growth factors are often produced by fibroblasts,nd the presence of a strong fibroblastic componentould activate the IGF-IR even in benign tumors. Fur-hermore, the IGF-IR is quasi-obligatory for transfor-ation, but, per se, is not an oncogene [1]: the impor-
ance of IGF-I (or IGF-II) in tumors is that it canccelerate their growth. For these reasons, an objectivetudy of the diagnostic and prognostic importance ofhis antibody must await an extended and exhaustivexamination of various forms of benign and malignantumors.
An antibody recognizing an activated PDGF recep-or has recently been published [60] and shown totain meningioma cells in humans. No extensivetudies on this antibody were presented in that pa-er. A similar antibody to the c-erbB-2 receptor wasescribed by Epstein et al. [61]. Antibodies that rec-gnized only an autophosphorylated IR were re-orted by Perlman et al. [62], although these authorsid not investigate the applicability of these antibod-es to sections of human tissues. Our antibody hasssentially the same properties as the antibody de-cribed by Shamah et al. [60] against the PDGFeceptor, but it is directed against the IGF-IR, whichlays a central role in the establishment and main-enance of the transformed phenotype [1].
In conclusion, we present here the development of anntibody that specifically recognizes an activated IGF-R, both in cells in tissue cultures and in sections ofuman cancers. This antibody should be very useful intudies of human cancers and their metastases. In areliminary survey, we have noticed that not all hu-
an breast cancers scored positive for the anti-pY1316ntibody. However, the eventual usefulness of this an-ibody can be determined only with careful prospectivetudies in which several sections from different areasf benign and malignant tumors can be examined.
This work was supported by Grants CA 56309 and CA 53424 fromhe National Institutes of Health.
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