Insulin and Insulin-like. Growth. Factor Signaling. Are Defective in the MDA MB-468. Human. Breast Cancer Cell Line'. Laura Sepp-Lorenzino,. Neal Rosen, and.
Vol.
5,
1077-
1083,
October
Cell
1994
Growth
& Differentiation
1077
Insulin and Insulin-like Growth Factor Signaling Are Defective in the MDA MB-468 Human Breast Cancer Cell Line’
Laura Sepp-Lorenzino, David E. Lebwohl2 Breast Cancer
Introdudion
and Gynecologic IL. S-L., D. E. L.I and Gastrointestinal Medicine Services, Division of Solid Tumor Oncology,
Department IL. S-L., New
Neal Rosen, and
York
of Medicine, N. RI,
Memorial
and
the Cell
Sloan-Kettering
Biology Cancer
and
IN.
Genetics Center,
Polypeptide growth factors regulate the proliferation of cancer cells. In particular, IGF3 types I and II are potent mitogens for breast cancer cell lines and may play an important role in the pathogenesis of human breast cancers (1-3). IGF-ll is expressed in stromal fibroblasts derived from breast tumors (4) and may therefore stimulate the proliferation of the malignant epithelial cells in a paracrine manner. Moreover, some breast cancer cell lines express IGF-II, suggest-
R.I
Program New
York,
10021
ing the
Abstract Polypeptide growth fadors including the insulin-like growth factors (IGFs), insulin, and transforming growth fador-a are mitogens for many breast cancer cell lines and may ad as regulators of cancer cell growth. In a human breast cancer cell line MCF-7, which expresses IGF-I receptor (IGF-IR), stimulation with insulin or lGFs resulted in autophosphorylation of the IGF-IR in an increased proportion of ras bound to GTP and in the association of phosphatidylinositol 3’-kinase (P13K) adivity and of p85-PI3K with Mr 185,000 phosphotyrosinylated proteins corresponding in size to insulin/IGF-IR substrates. These events were associated with enhanced proliferation. MDA MB-468 is a human breast cancer cell line which expresses insulin receptor and high levels of epidermal growth fador/transforming growth fador a receptor but low levels of IGF-IR. In this cell line, insulin stimulated autophosphorylation of IR at physiological concentrations and promoted the association of P13K adivity and of p85 with phosphotyrosine-containing proteins. Insulin did not, however, induce increased ras-GTP, and the cells exhibited minimal proliferation in response to insulin. Unlike insulin treatment, epidermal growth fador stimulation of MDA MB-468 cells is mitogenic and resulted in increased ras-GTP content, suggesting that the failure of insulin to induce these changes is not due to alterations in these signaling molecules. We conclude that there is a postreceptor defed in insulin signaling in MDA MB-468 which prevents the adivation of ras and the indudion of mitogenesis. Adivation of P13K by insulin is not sufficient to mediate mitogenesis. Alterations in the insulin/IGF pathway in MDA MB-468 may result from overexpression of epidermal growth fador receptor that occurs during tumor development and progression.
Received 5/18/94; revised 7/7/94; accepted 7/20/94. 1 Supported by Grants ROl CA 58706-01 and P20 Breast 2 To whom requests for reprints should be addressed, Medicine, Division of Solid Tumor Cancer Center, 1275 York Avenue,
Oncology, New York,
Memorial NY 10021.
SPORE CA-9i-33. at Department Sloan-Kettering
possibility
that
autocrine
IGF-II
may
allow
escape
from stromal influence (5). Consistent with this model, MCF-7 cells transfected with IGF-ll expression vectors acquire some aspects of the estrogen-independent phenotype (6, 7). IGF-lR content is elevated in most primary human breast cancers relative to normal breast tissue (8). Tumor development and progression, however, may also be associated with the amplification and overexpression of other receptor tyrosine kinases such as the EGFR or human
EGFR-2 (HER-2; Ref. 9). One hypothesis
is that overexpres-
sion of these receptors permits breast cancers to become independent of estrogen or of paracrine regulation by growth factors such as lGFs. IGFs I and II are similar in structure and effects to insulin (10). At physiological concentrations, lGFs I and II act mitogenically through the IGF-lR, while insulin acts through the IR. IGF-IR and IR are ligand-activated protein tyrosine kinases with highly homologous primary sequences (1 1), one of which has been cloned as IRS-i Their activation has similar consequences including activation of glucose transport (1 2, 1 3), induction of mitogenesis (1 3-1 5), and phosphorylation of a related set of endogenous substrates on tyrosine residues (1 6, 1 7), one of which has been cloned and designated as IRS-i (18). Several of the protein messengers involved in signal transduction by receptor protein tyrosine kinases have recently been identified (19, 20). Following ligand binding and kinase activation, a variety of signaling pathways become activated. Phosphotyrosinylated residues on the receptors themselves or on other protein substrates, such as IRS-i in the insulin and IGF pathways, serve as high-affinity .
binding sites for the recruitment
of 5H2-containing
proteins
such as phospholipase C--y, p1 20-rasGAP, p85-PI3K, SHC, and GRB2. Both GAP and GRB2 are involved in the regulation of ras activity, GAP by stimulating its intrinsic GTPase
of
activity,
and
change
factor
GRB2
protein
tyrosine
for
by binding
the guanine
ras, Sos (21-24). kinases
is required
nucleotide
ex-
Activation
of ras by
for the
proliferative
3 The abbreviations used are: IGF, insulin-like growth factor; EGFR, epidermal growth factor receptor; IGF-IR, IGF-l receptor; IRS, insulin/IGF-l receptor substrate(s); GAP, GTPase-activating protein; P13K, phosphatidylinositol 3’-kinase; DME:Fi2, Dulbecco’s modified Eagle’s medium: Ham’s F-i2 medium; FCS, fetal calf serum; NP-40, Nonidel P-40; SDS, sodium dodecyl sulfate; PBS, phosphate-buffered saline; HEPES, 4-(2-hydroxyethyl)-i -piperazineethanesulfonic acid; TLC, thin layer chromatography; IA, integrated absorbance; RT-PCR, reverse transcription-polymerase chain reaction.
1078
Defective
Insulin
Signaling
in MDA
MB-468
response to growth factors in many cell models (25, 26). Receptors may differ, however, in other aspects of signal transduction; in particular, insulin is known to activate P13K (27-29) and to increase rasGTP (30, 31 ) but not to phosphorylate or activate phospholipase C-y (32, 33). We studied the secondary messengers involved downstream from activated IR and IGF-IR in two breast cancer cells with different proliferative responses toward insulin. The cell line MCF-7 proliferates in response to insulin, while the MDA MB-468 line does not. In both lines, insulin activates a functional receptor kinase, IRS and P13K. Only in MCF-7, however, does insulin induce an increase in rasGTP. We observe, then, that there is a defect in the ability of insulin to exert a mitogenic signal in MDA MB468 that correlates with its inability to activate ras. As EGF is both mitogenic and activating of ras in this cell line, the lesion is particular to transduction of the signal elicited by insulin. Furthermore, insulin-induced association of P13K and IRS is not sufficient for induction of proliferation.
Results Insulin Activates of MCF-7 Cells.
L. Sepp-Lorenzino,
106
a)
E
z U
0
1
2
3
Time
(days)
4
5
6
B ras and P13K in the Proliferative
Response
The human breast cancer cell line MCF-7 was stimulated to proliferate in response to 1 M insulin in agreement with previous reports (Refs. 1 and 5; Fig. iA). We studied the action of insulin in these responsive cells, including receptor phosphorylation and activation of downstream signaling molecules. This cell line possesses predominantly IGF-IR and low levels of IR and EGFR (Fig. 1 CY IGF-lR autophosphorylation was studied directly in MCF-7 cells exposed to varying concentrations of insulin and lGFs. Maximal phosphorylation of IGF-lR occurred at 1 M insuhin, as well as by lower, more physiological concentrations of lGFs I and II (20 nM).4 Insulin-dependent phosphorylation of IGF-IR was greater than that by the IGFs, possibly due to modulation by IGF-binding proteins and by the IGF-Il receptor, both known to be expressed in this cell line (34, 35). Due to this effect, we decided to use 1 M insulin, which fully activated both the IGF-IR and IR, for the experiments described below. Next we studied activation of ras, which occurs in association with mitogenic activation of several receptor tyrosine kinases (25, 26). Insulin stimulation of fibroblast cells overexpressing human IR leads to a rapid but transient increase in rasGTP (30, 31). When MCF-7 cells were treated with insulin, a 3.5-fold increase in the proportion of rasGTP was detected as early as i mm after insulin challenge and returned to basal levels by 10 mm (Fig. 2, A and D). Similar results were observed in three independent experiments. The activation of P13K correlates with proliferation and transformation via tyrosine kinases (36). Stimulation of MCF-7 cells with 1 M insulin led to the association of P13K activity as well as of its regulatory subunit, p85-PI3K, with tyrosine phosphorylated proteins (Fig. 3, A and B, respectively). Maximal activity and association was attained at i mm. A broad band of phosphotyrosine-containing proteins in p85-PI3K immunoprecipitates was prominent in the region of Mr 1 85,000 (Fig. 3B), which corresponds in size to the known major substrates of IR and IGF-lR, IRS. In previous reports, phosphorylated IRS-i was shown to bind to p85-PI3K via phosphotyrosine-SH2 domain interactions
4
A
N. Rosen,
and
D.
E. Lebwohl,
unpublished
data.
MDA
FBS
MB-468
a) .0
E C
Insulin
a)
0
Control
0
2
4
8
6
Time
10
(days)
C lB aP-Tyr IPaIGF-IR
IPaIR
N-
N-
LLcx:
u-co
0(0
0(0
c;l-
/3 subunit Fig.
1.
Insulin
:
-
stimulates
MCF-7
human
breast
induces minimal proliferation in MDA MB-468 cells. MCF-7 cells were incubated in serum-free media with the addition of 1 M insulin 1
cancer
ja
cell
proliferation
but
cells. A, growth of MCF-7 media (control) or the same insulin) or 10% FCS. Cell
counts were determined on a hemocytometer at 2 and 5 days. Bars, SEM; no bars are shown if the SEM was
