The Journal of Clinical Investigation
Copyright © 2000 The American Society for Clinical Investigation,
Inc.

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Volume 105(1) January 2000 pp 9-13
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Anticancer drug targets: growth factors and growth factor signaling
[Perspective]
Gibbs,
Jackson B.

Section Editor(s): Kaelin,
William G. Jr.

WP16-101 Cancer Research,
Department of Cancer Research,
Merck Research
Laboratories,
Sumneytown Pike,
West Point,
Pennsylvania 19486,
USA.

Phone: (215) 652-5278; Fax: (215) 652-7320; E-mail: jay_gibbs@merck.com.

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Outline

Growth factor receptors
Targeting a GTPase switch
Inhibiting protein kinase effectors
Blocking lipid-mediated signaling
Conclusions
Section Description

Graphics

Table 1

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Signaling mechanisms that drive cell proliferation are closely associated with
tumor malignancy.

Components of these pathways,
encoded by some of the very
first oncogenes identified,
include the PDGF-like ligand Sis,
the tyrosine
kinases Src and HER-2/c-Neu (HER-2),
and the GTP-binding switch Ras.

The study
of communication by these oncoproteins has identified a complex array of
intracellular circuits.

In some cancers,
mutations in key components lead to
constitutive activation of these pathways; this activation is associated with
the proliferative properties of the tumor cells.

In this Perspective,
I provide
a broad overview of a growth factor signal transduction system,
with a focus on
those points that have been translated to drugs or clinical candidates.

Due to
editorial restrictions limiting the number of reference citations,
much of the
clinical data gleaned from abstracts is not listed in the references.

Instead,

the reader is directed to the 1999 Proceedings of the American Society of
Clinical Oncology and the 1999 Proceedings of the AACR-NCI-EORTC International
Conference.

Signaling pathways are initiated with the binding of a ligand,
such as PDGF,

EGF,
EGF-like ligands (e.

g.

, TGF-[alpha] and amphiregulin),
or IGF,
to its
cognate transmembrane receptor (1).

Ligand binding induces the dimerization of
receptor subunits,
promoting autophosphorylation of the receptor and recruiting
a variety of intracellular docking proteins (such as Grb2,
Shc,
and Nck) to the
plasma membrane.

These docking proteins create a molecular scaffold from which
subsequent signals emanate.

For example,
the guanine nucleotide exchange factor
Sos binds to Grb2,
which in turn interacts with the Ras protein.

Ras serves as a
molecular switch in the plasma membrane that alternates between an inactive
GDP-bound state and an active GTP-bound state.

Normally,
Ras is bound to GDP
because of the abundance of GTPase-activating protein and neurofibromin,
which
both suppress Ras function.

However,
upon recruitment of Sos to the membrane,

Sos binds Ras-GDP and facilitates release of GDP.

In cells,
the nucleotide GTP
is about 10-fold more abundant than GDP; GTP binds to Ras by mass action.

Ras-GTP adopts a conformation that permits interaction with down-stream targets
called effector molecules.

These effectors include the protein kinase Raf,
which
activates the MAP kinase cascade; GTPase-activating protein,
which links Ras to
the Rho/Rac pathway; and phosphoinositide (PI) 3'-kinase and Ral-guanine
nucleotide dissociation stimulator (Ral-GDS),
which activate lipid pathways (2).

The dysregulation of these signals in tumor cells leads to multiple cellular
changes,
including alterations in DNA synthesis,
lipid metabolism,
cellular
morphology,
cell adhesion properties,
and gene expression.

In the broadest sense,
the study of signaling mechanisms has already yielded
therapeutic agents in the treatment of cancer,
as evidenced by antiestrogens,

antiandrogens,
agonists of gonadotropin-releasing hormone,
and stem cell growth
factors,
for example.

However,
research into oncoproteins that function within
the signal transduction system is only beginning to be applied in the clinic.

Therapeutic approaches of interest include tools such as mAbs against the
extracellular domain of receptors,
oligonucleotides that are antisense to key
target proteins,
and small molecule inhibitors of enzymes (Table 1).

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Table 1 Examples of inhibitors of growth factor signaling for cancer treatment
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Growth factor receptors

Efforts to inhibit HER-2 yielded the first cancer therapeutic agent based on
research in growth factor signaling.

Unlike other members of the EGF receptor
family,
HER-2 has no known ligand (3).

HER-2 expression is upregulated in
approximately 25-30% of human breast cancers; this upregulation is believed to
promote HER-2 heterodimerization with other members of the EGF receptor family,

as well as HER-2 homodimerization,
which results in a constitutively active
tyrosine kinase.

Increased expression of HER-2 generally correlates with the
severity of disease,
and expression is consistently higher in tumor tissue than
in normal tissue,
making the tumor more prone to antibody therapy.

Genentech Inc.

developed the mAb trastuzumab,
which is directed against the
extracellular domain of HER-2 (4).

Use of this drug requires genotyping patient
tumor samples for the expression of HER-2.

It is thought that trastuzumab
inhibits the proliferation of breast cancer cells by several mechanisms (5).

First,
binding of trastuzumab is associated with upregulation of the p27Kip
inhibitor of some cyclin-dependent kinases.

Second,
this agent accelerates the
internalization and degradation of HER-2,
reducing the cellular level of
activated tyrosine protein kinase.

Third,
trastuzumab may induce immune-mediated
effects,
including cell-mediated cytotoxicity and complement fixation.

In
combination with cisplatin,
doxorubicin,
and especially paclitaxel,
trastuzumab
shows enhanced anti-tumor activity in preclinical models (6).

Trastuzumab has
also proved its value in the clinic and is particularly effective in combination
with paclitaxel (7,
8).

The combination of trastuzumab with doxorubicin also
appears to be effective,
but may have higher cardiotoxicity than trastuzumab
alone (8,
9).

>From the perspective of pharmaceutical development,
it is interesting to note
that the time from the discovery of the HER-2/c-neu oncogene in 1985 and the
association of HER-2 amplification in human breast cancer in 1987 to FDA
approval of trastuzumab in 1998 was a relatively short period.

This rapid
progress reflects an understanding of the underlying science,
as well as the
fact that trastuzumab is a biological agent.

In general,
biological agents may
be developed more quickly than are chemical entities.

Therapeutic antibodies have also been developed against the EGF receptor.

C225,

a human/mouse chimeric antibody (10),
and E7.

6.

3,
a fully human antibody (11),

bind to the EGF receptor extracellular domain and block EGF ligand binding.

These antibodies block the ligand-dependent proliferation of breast cancer cell
lines in cell culture,
and can induce tumor regression in mouse xenograft tumor
assays.

Like trastuzumab,
C225 appears to be especially effective in combination
with doxorubicin or paclitaxel (10).

C225 is currently undergoing clinical
evaluation.

In preliminary trial results,
complete responses were noted in head
and neck cancers when C225 was combined with radiotherapy.

The EGF receptor is also the target for the development of inhibitors of the
intracellular tyrosine kinase domain.

ZD-1839 and CP-358,
74,
competitive
inhibitors of ATP binding to the receptor's active site,
are currently in
clinical trials (12,
13).

Their mechanism of action has led to some concern
about safety,
given the variety and physiological significance of protein
kinases and other enzymes that bind ATP.

However,
these compounds appear to have
good anti-cancer activity in preclinical models,
with an acceptable therapeutic
index,
particularly in patients with non-small cell lung cancer.

The dermatological
toxicity observed for these drugs is most likely mechanism based,
arising as a
consequence of their intended biochemical activities.

More recently,
highly
potent and selective irreversible inhibitors of the EGF receptor kinase have
been reported,
such as PD-168,
93 (14).

This compound appears to bind specifically
to an active-site cysteine residue near the ATP binding site; its irreversible
binding may afford improved anti-tumor activity.

It will be interesting to
monitor the development of this class of inhibitor: such reactive molecules are
often dismissed as drugs,
because of their potential for nonspecific interactions,

but if they are sufficiently selective for their targets,
reactivity need not be
seen as a negative trait.

Aspirin,
for example,
is an irreversible inhibitor of
cyclooxygenases.

SU-101,
an inhibitor of PDGF receptor kinase activity (15,
16),
is currently in
phase II development for treating glioblastomas.

Another receptor tyrosine
kinase that has been explored with increasing attention as a drug target is the
IGF type I (IGF-I) receptor (17,
18).

This receptor activates cell proliferation,

but its role as an antiapoptotic signal may be more significant.

Initial
evidence from preclinical studies of an antisense oligonucleotide suggests that
IGF-I receptor inhibition can promote tumor apoptosis (17).

Targeting a GTPase switch

The ras gene,
discovered in 1978,
has attracted a great deal of attention
because it was the among the first oncogenes associated with human cancer,
and
studies of Ras function have helped to elucidate many of the mitogenic cell
signaling pathways (19).

Mutated forms of Kirsten-ras (Ki-ras) and N-ras are
found in solid tumors (lung,
colon,
pancreas,
and brain) and leukemias,
whereas
mutant Harvey-ras (Ha-ras) alleles are found in only a small subset of bladder,

head,
and neck tumors.

The agents currently in clinical trials that are based on
this area of research act either by regulating ras gene expression or by
inhibiting protein farnesylation.

An antisense oligonucleotide (ISIS-2503)
directed against Ha-ras expression (20) displayed significant anti-tumor
activity against a variety of human tumor cell lines in preclinical mouse tumor
xenograft studies.

ISIS-2503 appears to act against tumors whether or not they
have suffered mutations in Ha-ras,
but the basis of this broad activity is
unclear.

ISIS-2503 has completed phase I evaluation; an initial report noted
some disease stabilization when this agent was administered by continuous
intravenous infusion (20).

A second approach for inhibiting Ras function has attracted broad attention
within the pharmaceutical industry.

Ras proteins carry an essential lipid moiety
- a farnesyl group - at their COOH termini.

Genetic data indicate that
inhibition of Ras farnesylation blocks Ras localization to the plasma membrane.

Without this membrane localization,
Ras fails to interact with critical
regulatory and effector molecules (19),
and is transformation defective.

Hence,

farnesyl-protein transferase inhibitors (FTIs) are predicted to block cellular
transformation.

However,
the transferase reaction is essential not only to the
function of Ras,
but also to the function of at least 20 other farnesyl
proteins.

Thus,
FTIs are not truly Ras-specific inhibitors.

Nevertheless,
a
number of FTIs have been developed as potential anti-cancer drugs (21,
22).

Potent FTIs of diverse chemical structures inhibit tumor growth in both nude
mouse xenograft models and a variety of transgenic mouse tumor models -
including those that overexpress Ha-ras,
Ki-ras,
or N-ras (21).

The similar
effects of structurally distinct FTIs,
and their effectiveness at doses that
block substrate protein farnesylation,
confirm that these compounds achieve the
desired anti-tumor activity by inhibiting farnesyl-protein transferase.

Unlike
cytotoxic anti-tumor agents,
FTIs appear to act without overt systemic toxicity.

Since FTIs were originally thought to be cytostatic agents,
it was surprising to
observe in preclinical tissue culture and transgenic tumor models that they
induce apoptosis in tumor cells.

The induction of apoptosis occurs by caspase-3
activation and is independent of wild-type p53 function (21,
23) - an important
finding given the usual association of loss of p53 function with resistance to
chemotherapy (see Sellers and Fisher in this Perspective series).

In 1997 and 1998,
nearly 20 years after the discovery of Ras and about 9 years
after the discovery of Ras farnesylation,
clinical trials began with FTIs (22).

At least 4 different FTIs are currently undergoing evaluation: R115777;
SCH66336; L-778,
23; and BMS-214662 (24) (Table 1).

R115777 and SCH 66336 are
administered by the oral route,
L-778,
23 is given by continuous infusion,
and
BMS-214662 is administered either orally or intravenously.

The more advanced
trials with R115777 and SCH 66336 have reported dose-limiting toxicities
involving bone marrow and the gastrointestinal tract,
indicating that at high
enough concentrations,
FTIs can have general antiproliferative effects on normal
tissues.

The doses achieved in the clinic so far with L-778,
23 and SCH 66336
were sufficient to inhibit protein farnesylation in readily obtainable tissues
such as white blood cells and cells of the buccal mucosa.

Reports on the
efficacy of FTIs are anxiously awaited.

Based upon preclinical data,
it is
anticipated that FTIs will also be used in combination with other treatments,

such as paclitaxel,
vincristine,
cisplatin,
5-fluorouracil,
gemcitabine,

cyclophosphamide,
or radiation (25-28).

Inhibiting protein kinase effectors

A series of protein phosphorylation events within the cell ensue upon Ras
activation.

The first key step is the direct binding of the Raf protein kinase
to Ras-GTP (1,
2).

Raf in turn phosphorylates and activates MAP/Erk kinase
(MEK),
which in turn phosphorylates and activates MAP kinase.

The key role of
this pathway in Ras-mediated cellular transformation has inspired several
efforts to develop inhibitors of these protein kinase reactions (Table 1).

ISIS-5132,
an antisense oligonucleotide directed against Raf,
is in phase II
clinical development (20).

This compound causes a dose-dependent reduction of
c-Raf mRNA levels in preclinical tumor models.

This pharmacodynamic monitoring
has also been performed in the clinic using peripheral blood mononuclear cells
from treated patients as a tissue source.

In a phase I trial,
the median
reduction of Raf mRNA was 42% at 48 hours,
with significant inhibitions observed
up to 15 days,
although this decrease did not appear to be dose dependent.

Of
the 65 patients evaluated in these initial reports,
4 patients with ovarian,

pancreatic,
renal,
and colon cancer have seen their disease remain stable for up
to 10 months.

Interestingly,
in 2 of the other patients,
disease progression
coincided with the loss of suppression of Raf mRNA levels (20).

Raf protein kinase inhibitors remain at an earlier stage of development.

The
most extensive analysis is from Hall-Jackson et al.

(29,
30),
who characterized
the biological effects of both a direct Raf kinase inhibitor,
ZM 336372,
and a
p38 kinase inhibitor,
SB 203580,
which weakly inhibits Raf kinase activity.

Cells treated with ZM 336372 or SB 203580 exhibit a paradoxical increase in Raf
activity measured ex vivo,
indicating that these compounds do not inhibit Raf
signaling pathways.

ZM 336372 does not inhibit Ras- or Raf-mediated cellular
transformation,
but a preliminary report by Heimbrook et al.

(31) indicates that
the triarylimidazole derivative L-779,
50,
which inhibits Raf protein kinase
activity in vitro,
blocks intracellular signaling by Ki-Ras and Ha-Ras.

Two groups have recently described novel MEK inhibitors (Table 1).

Parke-Davis
Pharmaceutical Research,
which described the first MEK inhibitor,
PD098059,

identified a more potent and selective compound (PD-184352) from a coupled
biochemical screen that included GST-MEK,
MAP kinase,
and the MAP kinase
substrate myelin basic protein (32).

DuPont Pharmaceuticals Co.

identified U0126
in a cell-based assay that monitored AP-1 response elements,
and they subsequently
found that this compound inhibits MEK activity (33).

Neither PD-184352 nor U0126
compete for binding to ATP or protein substrates,
suggesting that these
compounds function as allosteric inhibitors of MEK.

Both compounds block MAP
kinase phosphorylation in cells,
and at doses that abolish intracellular MEK
activity,
PD-184352 inhibits the anchorage-independent growth of several human
tumor cell lines and causes cells to adopt a flattened morphology.

At similar
doses,
PD-184352 also inhibited tumor growth in mouse tumor xenograft models
(32).

The correlation between this surrogate biochemical endpoint and biological
activity provides strong evidence for mechanism-based anti-tumor activity,
but
MEK inhibitors remain at the preclinical development stage.

Blocking lipid-mediated signaling

Activation of growth factor receptors is also associated with changes in
phospholipid metabolism (1-3,
18).

In 1 pathway,
the phosphorylated residues on
the intracellular domain of these receptors bind phospholipase C,
which then
cleaves membrane phospholipids.

One of these breakdown products,
diacylglycerol,

can activate some forms of protein kinase C (PKC),
such as PKC-[alpha],
which
has been implicated in cell proliferative processes and tumorigenesis (34).

PKC-[alpha] expression has been found in some human breast tumors to be elevated
relative to surrounding normal tissue.

Both antisense inhibitors to PKC-[alpha]
(ISIS-3521) and inhibitors of PKC kinase activity (CGP 41251 and UCN-01) are in
clinical trials (Table 1).

The kinase inhibitors,
both of which are derivatives
of staurosporine,
potently inhibit PKC activity and are active in mouse tumor
xenograft models (34).

CGP 41251 also inhibits the P-glycoprotein transporter,

which mediates the multidrug resistance of many advanced tumors.

The toxicities
noted for UCN-01 and CGP 41251 in the clinic are so far not remarkable,
but this
may be related to the high capacity of these compounds to bind plasma proteins -
a characteristic that might also be expected to blunt their anti-tumor activity
(34).

The antisense compound ISIS-3521 exhibits an acceptable safety profile.

Its side effects - fatigue,
fever,
and thrombocytopenia - are typical of
phosphorothioate-based antisense compounds (20).

ISIS-3521 is being tested in
combination with carboplatin and paclitaxel in patients with non-small cell lung
cancer; preliminary data indicate partial responses in 6 of 8 patients treated.

In a second pathway,
activation of Ras directly activates PI 3'-kinase.

The
product of this reaction is then able to activate the protein kinase Akt,
which
is a suppressor of apoptosis (2).

Inhibition of PI 3'-kinase activity would then
be predicted to inactivate Akt activity and subsequently activate apoptotic
pathways in tumors.

In preclinical studies,
LY 294002 potently inhibited PI
3'-kinase.

This compound inhibits lipid signaling by growth factor receptors.

In
combination with an FTI,
it was shown to induce apoptosis in attached tumor
cells,
which normally do not respond to FTI alone (35).

This result raises the
interesting possibility that inhibitors of different steps of the signaling
pathways may be of greatest benefit when used in combination.

Conclusions

Growth factor-regulated proliferation pathways elucidated over the last 2
decades are finally reaching the clinic to be tested.

So far,
just 1 product,

trastuzumab,
has emerged,
but its apparent success provides much encouragement.

This product shows the therapeutic value of a treatment based upon a fundamental
genetic defect in a cancer and raises hopes for other agents,
such as those
summarized in Table 1.

It is interesting to note how our thinking has changed as
the basic research findings of growth factor signaling have been translated into
pharmaceutical entities.

First,
it has become clear that these compounds do not act solely on tumor
tissue.

Each agent has a particular toxicity that must be managed.

In some
cases,
as with EGF receptor inhibitors or FTIs,
these effects are mechanism
based,
but the undesirable consequences of other agents,
including phosphorothioate
antisense oligonucleotide compounds,
are structure based.

In either event,

therapies developed on growth signaling pathways offer new mechanisms to attack
cancer,
but they do not necessarily provide a true cure for cancer.

Second,
we have come to appreciate the value of combining these new inhibitors
with existing therapeutic regimens.

This realization reinforces the notion that
cancer is a disease of multiple and changing genetic alterations that must be
attacked with therapies having different mechanisms of action.

Therapies
designed based on knowledge of signal transduction pathways represent just 1
approach to developing new agents.

Clearly,
similar rational molecular
approaches for anti-cancer therapies may also be developed to control cell cycle
regulation and cell cycle checkpoints (see Shapiro in this Perspective series),

apoptosis (Sellers and Fisher,
this series),
telomere biology,
and angiogenesis
(Keshet,
this series).

How these different therapeutic strategies can best be
combined remains an open question.

Will it be better to have multiple inhibitors
targeting different steps of growth factor signaling pathways? Or will agents
directed at different fundamental aspects of a cancer cell prove the most
effective combination?

Finally,
it is interesting to note that surrogate pharmacodynamic endpoints are
beginning to be used for the development of signal transduction inhibitors (see
Druker and Lydon in this Perspective series).

In preclinical animal models,
the
biological efficacy of FTIs was monitored in relation to inhibition of protein
farnesylation and inhibition of downstream pathways such as MAP kinase and p70
kinase.

Likewise,
inhibition of MAP kinase phosphorylation showed a positive
correlation with the anti-tumor activity of the MEK inhibitor PD-184352.

Development of the ISIS antisense compounds has also been linked with a
reduction in the target mRNA levels.

This approach has also been carried into
the clinical development of some of these compounds,
such as has been reported
for SCH 66336,
L-778,
23,
and ISIS-5132.

Given the genetic complexities of
cancer,
it will be important to analyze whether monitoring these pharmacodynamic
endpoints provides useful clinical information,
particularly for compounds that
do not have clearly defined dose-limiting toxicities.

After all,
this is what
some believe to be the ultimate promise of these agents: lethality to tumors
without overt systemic toxicity.