(CANCER RESEARCH 58. 4250-4254. October I. 1998]

Advances in Brief

Enhanced Expression of the Insulin Receptor Substrate-2 Docking Protein inHuman Pancreatic Cancer1

Marko Konummu, Haruhisa Maruyama, Uwe Bergmann, l'ani Tangvoranuntakul, Hans G. Beger, Morris F. White,and Murray Korc2

Departments of Medicine, Biological Chemistry, and Pharmacology, University of California, ¡nine, California 92697 ¡Mu.K., H. M., P. T., Mu. K.I: Department of GeneralSurgery, University of (Jim, 89075 Ulm. Cermanv ¡U.B., H. G. BJ; and Joslin Diabetes Center and Department of Medicine, Han-ard Medical School, Boston, Massachusetts02215 ¡M.F. W.] '

Abstract

Insulin receptor substrate-2 (IRS-2) is a malusile docking protein

implicated in mitogenic signaling after activation of the insulin and insulin-like growth factor (IGF)-I receptors. In the present study, we characterized IRS-2 expression and function in human pancreatic cancer. IRS-2iiiKNA and protein were expressed in ASPC-1 and COLO-357 humanpancreatic cancer cell lines. Insulin, IGF-I, and IGF-II enhanced thegrowth of both cell lines, stimulated tyrosine phosphorylation of IRS-2,and increased IRS-2-associated phosphatidylinositol (PI) 3-kinase activity.The mitogenic effects of insulin, IGF-I, and IGF-II were markedly attenuated by the PI 3-kinase inhibitor LY 294002. Northern blot analysis of

total RNA extracted from normal and cancerous tissues revealed thatIRS-2 mRNA levels were increased in the cancer tissues i/' = 0.032). In the

normal pancreas, IRS-2 immunoreactivity was present at low levels in

some ductal and acinar cells and at moderate levels in a heterogeneouspattern in all of the endocrine islets. In the pancreatic cancers, IRS-2 wasabundant in the ductal-like cancer cells. These findings indicate that IRS-2

is overexpressed in human pancreatic cancer and suggest that it maycontribute to enhanced mitogenic signaling via the PI 3-kinase pathway,

thereby leading to excessive growth stimulation in this malignancy.

Introduction

Insulin and IGFs' are mitogenic polypeptides that bind to specific,

high-affinity receptors composed of two disulfide-linked extracellulara-subunits that contain the ligand binding sites and two /3-subunits

consisting of a transmembrane region and an intracellular regionexhibiting tyrosine kinase activity ( 1). Ligand binding to the insulin orIGF-IR results in activation of the intracellular receptor tyrosinekinase domains, subsequent auto-phosphorylation, and associationand tyrosine phosphorylation of the 1RS docking molecules (2-4).Pancreatic cancers overexpress IGF-I, IGF-IR, and IRS-1 (5, 6).

Moreover, in vitro studies have demonstrated that insulin and IGFs arepotent mitogens for cultured human pancreatic cancer cells (5-7).Despite the potential importance of IGF-I in human pancreatic cancer,nothing is presently known about the expression of IRS-2 in thismalignancy. Therefore, in this study, we compared IRS-2 expression

in normal and cancerous human pancreatic tissues and characterizedthe actions of IGF-I and related ligands on IRS-2 phosphorylation in

Received 7/10/98; accepted 8/17/98.The costs of publication of this article were defrayed in pan by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

' Supported by N1H Grant CA-40162 (to Mu. K.). Ma. K. was the recipient of

postdoctoral fellowship awards Ko 17I6/I-1 and 1-2 from the Deutsche Forschungsgemeinschaft. U. B. was the recipient of postdoctoral fellowship award Be 1762/1-1 fromthe Deutsche Forschungsgemeinschaft.

2 To whom requests for reprints should he addressed, at the Divison of Endocrinology.

Diabetes and Metabolism. Medical Sciences I, C240. University of California. Irvine, CA92697. Phone: (714) 824-6887; Fax; (714) 824-1035; E-mail: mkorc@uci.edu.

*The abbreviations used are: IGF, insulin-like growth factor; IGF-IR. IGF receptor I;

1RS, insulin receptor substrate; MTT. 3-(4.5-methylthiazol-2-yl)-2,5-diaphenyltertrazo-lium bromide; PI. phosphatidylinositol: PY20. antiphosphotyrosine: FBS. fetal bovineserum.

ASPC-1 and COLO-357 human pancreatic cancer cell lines. We nowreport that IRS-2 is overexpressed in human pancreatic cancer, whereit localizes in the cancer cells, and that insulin, IGF-I, and IGF-IIenhance IRS-2 tyrosine phosphorylation and IRS-2-associated PI3-kinase activity in cultured pancreatic cancer cells.

Materials and Methods

Materials. The following were purchased: ASPC-1 human pancreatic cancer cells from American Type Culture Collection (Manassas, VA); [y-32P]ATPand |a-"P]dCTP from Amersham Corp. (Arlington Heights, IL); PI substrate

from Avanti Polar Lipids Inc. (Alabaster, AL): insulin from Becton Dickinsonand Company (Franklin Lakes. NJ); LY 294002 from Biomol Research Laboratories Inc. (Plymouth Meeting, PA); BioMax ML and MS films from Kodak(Rochester. NY); silica gel 60 TLC plates from Merck (Darmstadt, Germany);immobilon-P nitrocellulose membranes from Millipore (Bredford, MA); en

hanced chemoluminescence substrate from Pierce Chemical Co. (Rocktord.IL); MTT, protein A-Sepharose, and secondary horseradish-conjugated anti-

rabbit antibody from Sigma Chemical Co. (St. Louis. MO); PY20 antibodyfrom Transduction Laboratory (Lexington, KY); IGF-II from United StatesBiochemical Co. (Cleveland. OH): rabbit polyclonal anti-IRS-2 antibodiesfrom Upstate Biotechnology Inc. (Lake Placid, NY). COLO-357 human pan

creatic cancer cells were a gift from R. S. Metzgar at Duke University(Durham, NC), and IGF-I was a gift from Genentech Inc. (South San Fran

cisco, CA).Cell Culture and Growth Assay. COLO-357 cells were grown in DMEM,

and ASPC-1 cells were grown in RPMI medium. All media were supplemented

with 8% FBS, penicillin G (100 units/ml), and streptomycin (100 /xg/ml). Cellswere maintained at 37°Cin humidified air with 5% CO,. Cell growth was

determined by the MTT colometric assay, as described previously (5—7).

Immunoblotting and Immunoprecipitation. Immunoblot analysis ofIRS-2 was carried out as described previously for IRS-1 (6. 7). Briefly, cells

were washed twice with ice-cold PBS and lysed in lysis buffer containing 50HIMTris (pH 7.5), 150 mm NaCl, 1% NP-40, 0.25% sodium deoxycholate. 2

mM EDTA, 50 mM NaF, 1 mM NaV,O4, 50 ¿ig/mlaprotinin. 10 /u.g/mlleupeptin. 10 /j.g/ml pepstatin A. 10 fig/ml benzamidine, and 1 mM phenyl-methylsulfonyl flouride. Cell lysates (30 fig) were subjected to 7% SDS-PAGEand electro-transferred to immobilon-P membranes. Membranes were blotted

with the indicated primary antibodies and corresponding secondary horseradish-conjugated antibodies as described (6. 7). Bound antibodies were visual

ized using enhanced chemoluminescence.For immunoprecipitation with IRS-2 antibodies, cells were grown to 50%

confluency in medium containing 8% FBS and then incubated for 24 h inserum-free medium containing antibiotics. 0.1% BSA. 5 mg/1 transferrin, and

5 fig/1 selenious acid, before stimulation with growth factors and cell lysis.Cell lysates (500 ¿igin 500 /¿Iof lysis buffer) were incubated for 2 h at 23°C

with IRS-2 antibodies (2 fig/sample), followed by a 1-h incubation withprotein A-Sepharose (30 /j.1) at 23°C.Precipitates were washed with ice-coldPBS, resuspended in loading buffer and boiled for 5 min at 100°C.After

centrifugation. the supernatants were subjected to Western blotting as described above. For reprobing, membranes were incubated for 30 min at 50°C

in buffer containing 2% SDS, 62.5 mM Tris (pH 6.7). and 100 mM 2-mercap-

toethanol.

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EXPRESSION OF IRS-2 IN HUMAN PANCREATIC CANCER

Northern Blot Analysis. Total RNA was extracted by the acid guani-dinium thiocyanate method, and poly(A)+ RNA was prepared by affinity

chromatography on oligo-dT cellulose (5). For Northern blotting, membranes

were hybridized under high stringency conditions with a 1.0 kb SacUNhelfragment of the mouse IRS-2 cDNA (8). cDNAs were labeled with[a-12P]dCTP (3000 Ci/mmol) by random priming before hybridization. A

human /3-actin cDNA and a mouse 7S cDNA were used as loading controls (5).The membranes were exposed to BioMax MS films at -80°C using intensi

fying screens.PI 3-kinase Activity Assay. PI 3-kinase activity was assayed in IRS-2

immunoprecipitates that were prepared as described above. Immunoprecipi-tates were sequentially washed with buffer A (PBS containing 1% NP-40 and

1 mM DTT), buffer B (100 mM Tris (pH 7.4), 0.5 M LiCl, and 1 mM DTT), andbuffer C (IO mM Tris, 100 mM NaCl, and 1 mM DTT) and resuspended andincubated for 5 min at 4°Cin 10 pii of buffer C (9). To assay for phospho-

rylation activity, 20 p.\ of PI substrate (0.5 mg/ml) were dispersed by sonica-

tion for 15 min in 50 mM HEPES (pH 7.6), 1 mM EGTA, and 1 mM NaH2PO4before use and added to the buffer solution. After a 5-min incubation at 23°C,

the reaction was started by the addition of 10 fM.1of 5 X ATP solution (50 mMMgCl2, 100 mM HEPES, and 250 /U.MATP including 10 nCi/jul [y-32P]ATP).

The reaction was stopped after 5 min by adding 4 N HC1 (15 ¿¿1).To separatethe phospholipids, the solution was vortexed after the addition of 130 ^1 ofchloroform:methanol (1:1). The phospholipid containing chloroform phase (35/A!) was spotted onto TLC plates coated with 1% potassium oxalate andactivated at 110°Cfor 90 min. Phosphorylated products were separated in

chloroform:methanol:NH4OH:H2O (600:470:20:113) until the solvent reachedthe top of the plates. The dried plates were exposed to Kodak Biomax Lightfilms at -80°C using intensifying screens.

Patients and Tissue Samples. Normal human pancreatic tissue sampleswere obtained through a donor program. There were six female and eight maleorgan donors with a median age of 26 years (range, 2-54). Pancreatic adeno-

carcinoma tissues were obtained from 10 female and 6 male patients having amedian age of 64 years (range, 44-77). All cancer patients underwent surgery

for pancreatic cancer as recently described (10). Samples were either immediately frozen upon surgical removal in liquid nitrogen and stored at -80°Cuntil RNA extraction, or fixed in Bouin's solution for 18-20 h and embedded

in paraffin for histológica! analysis. Studies involving human tissues wereapproved by the Ethics Committee of the University of Ulm and the HumanSubjects Committee of the University of California.

Immiinohistochemistry. Paraffin-embedded 4-/im tissue sections wereimmunostained using the streptavidin-peroxidase technique, as described pre

viously by Kornmann et al. (10). After deparaffinization and blocking endogenous peroxidase activity, the sections were incubated for 15 min at 23°Cwith10% normal goat serum and for 16 h at 4°Cwith a rabbit polyclonal antibody

against IRS-2 (0.5 ng/ml) that was also used for immunoblotting. Bound

antibodies were detected with biotinylated goat antirabbit IgG secondaryantibodies and streptavidin-peroxidase complex, using diaminobenzidine tet-rahydrochloride as the substrate. Sections were counterstained with Mayer's

hematoxylin. Omission of primary antibodies or incubation in the presence ofnonimmunized rabbit IgG instead of primary antibodies did not yield anyimmunoreactivity.

Statistics. Statistical analysis was performed with SigmaStat software (Jan-

del Scientific, San Raphael, CA). The Rank Sum Test was used and a P <0.05

was taken as the level of significance.

Results

IRS-2 expression in ASPC-1 and COLO-357 cells was characterized by immunoblotting using highly specific rabbit polyclonal antibodies against IRS-2 (11) and by Northern blotting using a 1.0-kbmouse IRS-2 cDNA fragment (3, 8). The bands representing the Mr190,000 IRS-2 protein and the 7.2-kb IRS-2 mRNA transcript (3)were readily evident in both cell lines (Fig. 1, A and B). The effectsof insulin, IGF-I, and IGF-II on tyrosine phosphorylation of IRS-2 andIRS-2-associated PI 3-kinase activity were investigated next. Analysisof the tyrosine phosphorylation status of the IRS-2 immunoprecipitates with PY20 antibodies revealed that insulin and IGF-I enhancedtyrosine phosphorylation of IRS-2 in ASPC-1 cells, whereas IGF-II

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Fig. 1. Effects of growth factors on lyrosine phosphorylation of IRS-2 and PI 3-kinaseactivity in ASPC-1 and COLO-357 cells. A. IRS-2 protein expression. Immunoblotanalysis of total cell lysates (30 fig/lane) was carried out using a highly specific anti-IRS-2antibody (0.5 /ig/ml). B, IRS-2 mRNA expression. Northern blol analysis of poly(A)*

RNA (2 fig/lane) was carried out using a 1-kb mouse IRS-2 cDNA fragment thaicross-hybridizes with human IRS-2 (250.000 cpm/ml). A human ß-actincDNA fragment

(25,000 cpm/ml) was used as loading control. Molecular weigh! and rRNA markers areindicated on the left. C and D. tyrosine phosphorylation of IRS-2. Quiescent ASPC-1 (Oand COLO-357 (D) cells were treated for 5 min with 5 nM of the indicated ligands beforecell lysis. After immunoprecipitation (/PI of total cell lysates (500 fig) with anti-IRS-2antibodies (2 pig/sample), samples were subjected to immunoblot (IB) analysis with PY20antibodies (0.2 (¿g/rnl:'o/1) and anti-IRS-2 antibodies (0.5 fig/ml: hniinm). E and I-', IRS-2

associated PI 3-kinase activity. IRS-2 immunocomplexes from ASPC-1 (E) and COLO-357 (F) cells were incubated with PI substrate in the presence of [•y-12P]ATPandseparated by TLC. Plates were exposed lo BioMax ML film for l h al -80°C. Phospho-

rylaled PI and the origin of the samples are indicated on the left.

was without effect (Fig. 1C). In contrast, in COLO-357 cells, all threegrowth factors enhanced IRS-2 tyrosine phosphorylation (Fig. ID). Inboth cell lines, the magnitude of this stimulatory effect was greatestwith insulin. A PI 3-kinase activity assay after IRS-2 immunoprecipitation revealed that all three growth factors enhanced PI 3-kinaseactivity in both cell lines (Fig. 1, E and F). However, the effect ofIGF-II on PI 3-kinase activity in ASPC-1 cells was relatively modest.

To further assess the role of PI 3-kinase activation in insulin- andIGF-mediated mitogenesis in ASPC-1 and COLO-357 cells, we nextcompared the growth stimulatory effects of these ligands in theabsence or presence of the selective PI 3-kinase inhibitor LY 294002at concentrations that did not inhibit basal growth. Insulin, IGF-I, andIGF-II (5 nin each) enhanced the growth of ASPC-1 and COLO-357

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EXPRESSION OF IRS-2 IN HUMAN PANCREATIC CANCER

cells (Fig. 2), and this effect was significantly attenuated by LY294002 (Fig. 2).

Northern blot analysis revealed the presence of variable levels ofthe 7.2-kb IRS-2 mRNA transcript (3) in normal and cancerouspancreatic tissues (Fig. 3A). After prolonged exposure of the autora-diographs, the IRS-2 mRNA transcript was detectable in all normal

and cancerous tissue samples. Densitometric analysis with normalization to 7S indicated that the median level of IRS-2 mRNA wasincreased by 1.4-fold in the cancer tissues in comparison with themedian level in the normal pancreatic samples (P = 0.032). Overall,4 of the 16 cancer samples displayed IRS-2 mRNA levels that clearlyexceeded the IRS-2 mRNA levels observed in the 12 normal samples

tested (Fig. 3B).Immunohistochemical analysis using the same highly specific anti-

IRS-2 antibody that was used for immunoblotting was performednext. In the normal pancreas, faint IRS-2 immunoreactivity was

present in some ductal and acinar cells (Fig. 4, A and B), and moderateIRS-2 immunoreactivity was present in at least some islet cells in all

of the endocrine islets (Fig. 4A and B, arrowheads). In the pancreaticcancer samples, a similar pattern of IRS-2 immunoreactivity was seen

in the endocrine islets adjacent to the pancreatic cancer cells (data notshown). In addition, moderate to strong IRS-2 immunoreactivity waspresent in many of the ductal-like cancer cells (Fig. 4, C and D).

300

100

300

Insulin IGF-I IGF-II

100-\Insulin IGF-I IGF-II

Fig. 2. Effects of insulin. IGF-I. IGF-II. and LY 294002 on pancreatic cancer cellgrowth. A, ASPC-I cells (20.000 cells/well) were seeded in 96-well plates, incubated for24 h in medium containing S'/i FBS and then for 48 h in medium containing antibiotics.

0.1% BSA, 5 mg/1 transferrin, and 5 /Ag/l selenious acid in the presence of 5 nvi of theindicated ligands alone (D) or together with 3 ^M of LY 294002 (0. Cell growth was thendetermined using the MTT assay. Results are expressed as the percentage of growth whencompared with untreated controls and are the means (±SE) of quadruplicate determinations from three separate experiments. *, P ^0.0009 compared with untreated controls;**. P £0.0009 compared with ligand without LY 294002. B. COLO-357 cells weretreated as described in A, except that the concentration of LY 294002 was 5 /¿M.»,P<0.005 compared with untreated controls; »*,P <0.007 compared with ligand withoutLY 294002.

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Normal CancerFig. 3. IRS-2 mRNA expression in pancreatic tissues. A. Northern blot analysis of total

RNA (20 /ig/lane) from six normal and nine pancreatic cancer samples was carried out asdescribed in the legend to Fig. Iß.The 7S cDNA was used as loading control. Themigration site of the 28S rRNA is indicated on the left. B. comparison of the relative IRS-2mRNA expression in normal and cancerous (n = 16) pancreatic tissues. Autoradiographsfor IRS-2 and 7S were scanned and analyzed by densitometry. The ratio of IRS-2 and 7S(arbitrary units) was then plotted for normal (»= 14) and cancerous (n = 16) samples.Pancreatic cancers displayed a higher median IRS-2 expression {dashed lines} than thenormal samples (P = 0.032).

Discussion

IRS-1 and -2 are homologous docking proteins that mediate theactions of IGF-I and insulin (4, 12). The presence of over 30 unique

phosphorylation motifs on each of the two 1RS proteins enables themto interact with a variety of down-stream signaling molecules (4). Inaddition to their importance for insulin/IGF-I signaling, 1RS proteins

contribute to the signal transduction of several cytokine receptors (4,13), underscoring the potential importance of these docking moleculesfor mediating intracellular signaling of a variety of extracellularstimuli.

Previously, we reported that IRS-1 is overexpressed in humanpancreatic cancer (6). In the present study, we determined that IRS-2

is also overexpressed in this malignancy. Although there was only a40% increase in IRS-2 mRNA levels in the pancreatic cancers, this

increase was statistically significant by comparison with the levelsobserved in the normal pancreas. By immunohistochemistry, IRS-2

was consistently present in the endocrine islets in the normal pancreas,raising the possibility that IRS-2 may participate in the regulation ofendocrine functions in the human pancreas. In addition, faint IRS-2

immunoreactivity was present in some ductal and acinar cells. Theseobservations suggest that a significant component of the IRS-2 signal

observed by Northern blotting of normal pancreas RNA derives fromboth the endocrine and exocrine cells. In contrast, only faint IRS-2

immunoreactivity was evident in the abundant stromal elements in the4252

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EXPRESSION OF IRS-2 IN HUMAN PANCREATIC CANCER

Fig. 4. Localization of IRS-2 protein in pancreatic tissues. Immunohistochemistry with highly specific anti-IRS-2 antibodies (0.5 fig/ml) that were also used for Western blot analysisrevealed that IRS-2 was principally present in the islet cells (arrowheads) and also in some ductal and acinar cells in the normal pancreas tA and lì).In the pancreatic cancer samples.IRS-2 was abundant in the cancer cells (C and D). Each panel derives from a different pancreatic tissue sample. Bar. 25 ¿un.

pancreatic cancer samples, whereas a strong IRS-2 signal was present

in the pancreatic cancer cells. Taken together, these findings suggestthat the pancreatic cancer cells within the tumor mass express higherlevels of IRS-2 than indicated by Northern blotting.

Several lines of evidence from our in vitro studies suggest thatIRS-2 contributes to enhanced mitogenic signaling in pancreaticcancer. First, IRS-2 was expressed in both tested pancreatic cancercell lines. Second, insulin, IGF-I, and IGF-II enhanced tyrosinephosphorylation of IRS-2 and IRS-2-associated PI 3-kinase activity in COLO-357 cells, and IGF-I and insulin exerted similareffects in ASPC-1 cells. Inasmuch as IGF-II did not enhancetyrosine phosphorylation of IRS-2 in ASPC-1 cells and only minimally stimulated IRS-2-associated PI 3-kinase activation in thesecells, it is likely that IGF-II enhances the growth of these cells via

a different mechanism. Third, in both cell lines inhibiting PI3-kinase activity with concentrations of LY 294002 that did not

alter basal growth markedly attenuated the effect of all threegrowth factors on mitogenesis. Taken together, these observationssuggest that IRS-2 is an important mediator of mitogenic signaling

in pancreatic cancer cells, and that this effect is mediated, in part,via PI 3-kinase.

Overexpression of IRS-1 results in an increase in mitogenic response to insulin and IGF-I (14-16), whereas a decrease in IRS-1

levels diminishes this response (17, 18). Despite the 43% amino acidsequence homology between IRS-2 and IRS-1 (4), these molecules

may mediate distinct signals. Thus, studies with knockout mice suggest that IRS-1 may best mediate the biological actions of IGF-I,

whereas IRS-2 may preferentially mediate the activation of insulin on

glucose transport (3, 19). In contrast, studies with cultured cell lineshave suggested that IRS-2 may be better adapted to mediate mitogenicsignaling than IRS-1 (8). However, when IRS-1 or IRS-2 were expressed in IRS-1-deficient cells, both proteins wore required for a

complete mitogenic response, indicating that they are not functionallyinterchangeable with respect to mitogenesis (20). Furthermore, inestrogen receptor positive breast cancer cells 1RS-1 is the majorsignaling molecule activated by insulin and IGF-I (21), whereas inRINmSF insulin-secreting cells IRS-2 seems to mediate the actions ofIGF-I on mitogenesis (22). We have previously shown that IGF-Ienhances tyrosine phosphorylation of IRS-1 in A^PC-1 and COLO-357 cells (7). Thus, the concomitant overexpression of IRS-1 (6) andIRS-2 (present findings) in human pancreatic cancer underscores theirpotential importance in conferring a growth advantage onto pancreatic-

cancer cells.Previous studies have demonstrated that pancreatic cancers over-

express IGF-I and IGF-IR (5). Although insulin and IGF-II are not

overexpressed in this malignancy, both arc present in endocrine isletcells, which are dispersed throughout the exocrine pancreas (23).Furthermore, as a consequence of the existence of an intrapancrcaticportal circulation, portions of the exocrine pancreas are exposed tohigh levels of islet cell hormones (23). This anatomical arrangementmay allow for islet cell hormones to act via a so-called proxicrine

mechanism on pancreatic exocrine cells, including cancer cells (23).Inasmuch as insulin and IGF-II bind and activate the IGF-IR (12). our

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EXPRESSION OF IRS-2 IN HUMAN PANCREATIC CANCER

findings suggest that there is a potential for IGF-I, insulin, and IGF-II

to excessively activate mitogenic signaling in pancreatic cancer cellsby acting via autocrine, paracrine, and proxicrine mechanisms on theoverexpressed IGF-IR, IRS-1, and IRS-2. Therefore, pharmacological

or genetic interference with this pathway may have an importanttherapeutic potential in pancreatic cancer.

Acknowledgments

We thank R. S. Metzgar at Duke University (Durham, NC) for the gift ofCOLO-357 human pancreatic cancer cells and Genentech Inc. (South SanFrancisco, CA) for the gift of human recombinant IGF-I.

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