"this" "is about" "Her2Neu"
"J Clin Invest, Volume 105(1).January 2000.9-13." "is citation for" "this"

The American Society for Clinical Investigation, Inc.

Volume 105(1) January 2000 pp 9-13 -

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.

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

"Signaling mechanisms" "drive" "cell proliferation"
"Signaling mechanisms" "are 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.

"Components of these pathways" "include" "the PDGF-like ligand Sis"
"Components of these pathways" "include" "the tyrosine kinases Src"
"Components of these pathways" "include" "HER-2/c-Neu (HER-2)"
"Components of these pathways" "include" "the GTP-binding switch Ras"

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

"Oncoprotein communication studies" "have 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.

"Mutations in key components" "lead to constitutive activation" "of these pathways"
"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.

"Perspective" "provides a broad overview" "of a growth factor signal transduction system"
"Focus" "is on points translated" "to drugs"
"Focus""is on points translated" "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.

"Clinical data 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.

"The reader" "is directed to" "the 1999 Proceedings of the American Society of Clinical Oncology"
"The reader" "is directed to" "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).

"Signaling pathways" "are initiated with the binding" "of PDGF" "to its cognate transmembrane receptor"
"Signaling pathways" "are initiated with the binding" "of EGF" "to its cognate transmembrane receptor"
"Signaling pathways" "are initiated with the binding" "of "EGF-like ligands TGF-[alpha]" "to its cognate transmembrane receptor"
"Signaling pathways" "are initiated with the binding" "of "EGF-like ligands amphiregulin" "to its cognate transmembrane receptor"
"Signaling pathways" "are initiated with the binding" "of IGF" "to its cognate transmembrane receptor"

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.

"Ligand binding" "induces" "dimerization of receptor subunits"
"Dimerization of receptor subunits" "promotes" "autophosphorylation"
"Dimerization of receptor subunits" "recruits" "intracellular docking protein Grb2" "to the plasma membrane"
"Dimerization of receptor subunits" "recruits" "intracellular docking protein Shc" "to the plasma membrane"
"Dimerization of receptor subunits" "recruits" "intracellular docking protein 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.

"Sos" "binds to" "Grb2"
"Grb2" "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.

"Ras" "serves as" "a molecular switch" "in the plasma membrane"
"Ras" "alternates between" "an inactive GDP-bound state"
"Ras" "alternates between" "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.

"Ras" "is bound to" "GDP"
"Ras" "binds because of" "the abundance of GTPase-activating protein"
"Ras" "binds because of" "neurofibromin"
"GTPase-activating protein" "suppress" "Ras function"
"Neurofibromin" "suppress" "Ras function"

However, upon recruitment of Sos to the membrane, Sos binds Ras-GDP and facilitates release of GDP.

"Sos" "is recruited to" "the membrane"
"Sos" "binds" "Ras-GDP"
"Sos" "facilitates release of" "GDP"

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

"GTP" "is about" "more abundant than GDP"
"GTP" "binds to" "Ras"
"GTP" "binds" "by mass action"

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

"Ras-GTP" "adopts" "a conformation"
"Conformation" "permits interaction with" "down-stream targets"
"Down-stream targets" "are 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).

"Effectors" "include" "the protein kinase Raf"
"Effectors" "include" "GTPase-activating protein"
"Effectors" "include" "phosphoinositide (PI) 3'-kinase"
"Effectors" "include" "Ral-guanine nucleotide dissociation stimulator (Ral-GDS)"
"Kinase Raf" "activates" "the MAP kinase cascade"
"GTPase-activating protein" "links Ras to" "the Rho/Rac pathway"
"Phosphoinositide (PI) 3'-kinase" "activates" lipid pathways
"Ral-guanine nucleotide dissociation stimulator (Ral-GDS)" "activates" "lipid pathways"

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.

"Signal dysregulation" "in tumor cells" "leads to" "multiple cellular changes"
"Cellular changes" "include" "alterations in DNA synthesis"
"Cellular changes" "include" "alterations in lipid metabolism"
"Cellular changes" "include" "alterations om cellular morphology"
"Cellular changes" "include" "alterations in cell adhesion properties"
"Cellular changes" "include" "alterations in 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.

"Study of signaling mechanisms" "has yielded" "therapeutic agents in the treatment of cancer"
"Antiestrogens" "are" "therapeutic agents"
"Antiandrogens" "are" "therapeutic agents"
"Agonists of gonadotropin-releasing hormone" "are" "therapeutic agents"
"Stem cell growth factors" "are" "therapeutic agents"

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

"Research" "is investigating" "oncoproteins"
"Oncoproteins" "function within" "the signal transduction system"
"Research" "isbeing applied" "clinicaly"

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

"Therapeutic approaches" "include "mAbs"
"mAbs" "are used against" the extracellular domain of receptors"?
"Therapeutic approaches" "include "oligonucleotides"
"Oligonucleotides" "are antisense to" "key target proteins"
"Therapeutic approaches" "include "small molecule inhibitors of enzymes"

(Table 1). ---------------------------------------------- Table 1

Examples of inhibitors of growth factor signaling for cancer treatment ----------------------------------------------

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

"Growth factor signaling research" "attempted to inhibit" "Her-2"
"Inhibited HER-2" "yielded" "first cancer therapeutic agent"

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

"HER-2c" "has" no known ligand"
"Other "EGF receptors" "have" "ligands"

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.

"HER-2 expression" "is" "upregulated"
"Upregulation" "occurs in" "approximately 25-30% ofhuman breast cancer"
"Upregulation" "is believed to promote" "HER-2 heterodimerization"
"HER-2 heterodimerization" "occurs with" "other members of the EGF receptor family"
"HER-2 homodimerization" "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.

"HER-2 overexpression" "correlates with" "the severity of disease"
"HER-2 overexpression" is "consistently higher" "in tumor tissue"
"HER-2 expression" "is not higher" "in normal tissue"
"Tumors" "are more prone to" "antibody therapy"

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

"Genentech Inc." "developed" "the mAb trastuzumab"
"mAb trastuzumab" "is directed against" "the extracellular domain of HER-2"

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

"This drug" "requires genotyping" "patient tumor samples"
"Patient tumor samples" "are genotyped" "for the expression of HER-2"

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

"Trastuzumab" "may inhibit" "proliferation of breast cancer cells"
"Trastuzumab" "may inhibit" "by several mechanisms"

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

"Trastuzumab binding" "is associated with" "upregulation" "of the p27Kip inhibitor"
"p27Kip inhibitor" "is associated with" "some cyclin-dependent kinase"

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

"Trastuzumab" " accelerates" "internalization of HER-2"
"Trastuzumab" " accelerates" "degradation of HER-2"
"Internalization of HER-2" "reduces" "cellular level of activated tyrosine protein kinase"
"degradation of HER-2" "reduces" "cellular level of activated tyrosine protein kinase"

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

"Trastuzumab" "may induce" "immune-mediated effects"
"Immune-mediated effects" "include" "cell-mediated cytotoxicity"
"Immune-mediated effects" "include" "cell-mediated complement fixation"

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

"Trastuzumab with cisplatin" "shows" "enhanced anti-tumor activity in preclinical models"
"Trastuzumab with doxorubicin" "shows" "enhanced anti-tumor activity in preclinical models"
"Trastuzumab with paclitaxel" "shows" "enhanced anti-tumor activity in preclinical models"

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

"Trastuzumab" has proven" "its value in the clinic"
"Trastuzumab" "is particularly effective" "in combination with paclitaxel"

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

"Trastuzumab with doxorubicin" "appears to be" "effective"
"Trastuzumab with doxorubicin" "may have higher cardiotoxicity" "than trastuzumab alone"

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.

"The HER-2/c-neu oncogene" "was discovered in" "1985"
"The HER-2/c-neu oncogene" "was associated with" "HER-2 amplification in human breast cancer in 1987"
"The FDA" "approved" "trastuzumab in 1998"

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

"This rapid progress" "reflects an understanding of" "the underlying science"
"Trastuzumab" "is" "a biological agent"

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

"Biological agents" "may be developed more quickly" "than chemical entities"

Therapeutic antibodies have also been developed against the EGF receptor.

"Therapeutic antibodies" "have 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.

"C225" "is" "a human/mouse chimeric antibody"
"E7.6.3" "is" "a fully human antibody"
"C225" "binds to" "the EGF receptor extracellular domain"
"C225" "blocks" "EGF ligand binding"
"E7.6.3" "binds to" "the EGF receptor extracellular domain"
"E7.6.3" "blocks" "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.

"These antibodies" "block" "the ligand-dependent proliferation of breast cancer cell lines in cell culture"
"These antibodies" "can induce" "tumor regression"
"Tumor regression" "is" "in mouse xenograft tumor assays"

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

"C225" "is similar to" "trastuzuma"
"C225 with doxorubicin" "appears to be" "especially effective"
"C225 with paclitaxel" "appears to be" "especially effective"

C225 is currently undergoing clinical evaluation.

"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.

"Preliminary trials "were" "held"
"C225 with radiotherapy"" prompted" "complete responses"
"Complete responses" "were noted" "in head and neck cancers"

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

"The EGF receptor" "is targeted for" "development of inhibitors"
"Inhibitors" "are" "of the intracellular tyrosine kinase domain"

ZD-1839 and CP-358,774, competitive inhibitors of ATP binding to the receptor's active site, are currently in clinical trials (12, 13).

"ZD-1839" "is" "a competitive inhibitors of ATP binding"
"CP-358,774" "is" "a competitive inhibitors of ATP binding"
"Binding" "occurs at" "the receptor's active site"
"ZD-1839" "is currently in" "clinical trials"
"CP-358,774" "is currently in" "clinical trials"

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.

"ZD-1839's mechanism of action" "has led to" "some concern about safety"
"CP-358,774's mechanism of action" "has led to" "some concern about safety"
"A variety of protein kinases and other enzymes" "bind" "ATP"
The 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.

"These compounds" "appear to have" "good anti-cancer activity in preclinical models"
"These compounds" "appear to have" "an acceptable therapeutic index"
"The therapeutic index" "appears good" "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.

"Dermatological toxicity" "is most likely" "mechanism based"
"Dermatological toxicity" "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-168,393 (14).

"Highly potent EGF receptor kinase inhibitors" "have been reported" such as PD-168,393 (14).
"Selective irreversible EGF receptor kinase inhibitors" "have been reported"
"PD-168,393" "is" "a highly potent and selective irreversible EGF receptor kinase inhibitor"

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.

"This compound" "appears to specifically bind to" "an active-site cysteine residue"
"The active-site cysteine residue" "is near" "the ATP binding site"
"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.

"Reactive molecules" "are often dismissed" "as drugs",
"Reactive molecules" "have potential for nonspecific interactions"
"Reactive molecules" "can be selective for" "their targets"
"Reactivity" "need not be seen as" "a negative trait"

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

"Aspirin" "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.

"SU-101" "is" "an inhibitor of PDGF receptor kinase activity"
"SU-101" "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).

"The IGF type I (IGF-I) receptor" "has been explored as" "a drug target"
"IGF type I (IGF-I) receptor" "is a" tyrosine kinase"

This receptor activates cell proliferation, but its role as an antiapoptotic signal may be more significant.

"IGF type I (IGF-I)" "activates" "cell proliferation"
"IGF type I (IGF-I)'s role" "is as" "an antiapoptotic signal"
"IGF type I (IGF-I)'s role" "may be" "more significant"

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

"Preclinical studies" "have been conducted" "on an antisense oligonucleotide"
"Studies" suggests" "IGF-I receptor inhibition"
"IGF-I receptor inhibition" "can promote" "tumor apoptosis"

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).

"The ras gene" "was discovered in" "1978"
"The ras gene" "has attracted" "a great deal of attention"
"The ras gene" "was the among" "the first oncogenes associated with human cancer"
"Studies of Ras function" "have helped to elucidate" "many of the mitogenic cell signaling pathways"

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.

"Mutated forms of Kirsten-ras (Ki-ras)" "are found in" "solid tumors"
"Mutated forms of Kirsten-ras (Ki-ras)" "are found in" "lung"
"Mutated forms of Kirsten-ras (Ki-ras)" "are found in" "colon"
"Mutated forms of Kirsten-ras (Ki-ras)" "are found in" "pancreas"
"Mutated forms of Kirsten-ras (Ki-ras)" "are found in" "brain"
"Mutated forms of Kirsten-ras (Ki-ras)" "are found in" "leukemias"
"Mutated forms of N-ras" "are found in" "solid tumors"
"Mutated forms of N-ras" "are found in" "lung"
"Mutated forms of N-ras" "are found in" "colon"
"Mutated forms of N-ras" "are found in" "pancreas"
"Mutated forms of N-ras" "are found in" "brain"
"Mutated forms of N-ras" "are found in" "leukemias"
"Mutant Harvey-ras (Ha-ras) alleles" "are found in" "only a small subset of bladder tumors"
"Mutant Harvey-ras (Ha-ras) alleles" "are found in" "only a small subset of head tumors"
"Mutant Harvey-ras (Ha-ras) alleles" "are found in" "only a small subset of 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.

"Agents" "are currently in" "clinical trials"
"Agents" "act by regulating" "ras gene expression"
"Agents" "act 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.

"Antisense oligonucleotide (ISIS-2503)" "is directed against" "Ha-ras expression"
"Antisense oligonucleotide (ISIS-2503)" "displayed" "significant anti-tumor activity"
"Significant anti-tumor activity" "was against" "a variety of human tumor cell lines"
"Results" "were 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" "appears to act against" "tumors"
"Tumors" "may have suffered" "Ha-ras mutations"
"Tumors" "may not have suffered" "Ha-ras mutations"
"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).

"ISIS-2503" "has completed" "phase I evaluation"
"Initial report" "notes" "some disease stabilization"
"ISIS-2503" "was administered by" "continuous intravenous infusion"

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

"The pharmaceutical industry" "is interested in" "a second approach for inhibiting Ras function"

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

"Ras proteins" "carry" "an essential lipid moiety"
"This essential lipid moiety" "is" "a farnesyl group"
"This essential lipid moiety" "is carried at" "their COOH termini"

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

"Genetic data" "indicate blockage of" "Ras localization"
"Ras farnesylation inhibition" "blocks" "Ras localization"
"Ras localization" "is blocked at" "the plasma membrane"

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

"Membrane localization failure" "causes" "Ras failure"
"Ras" "fails to interact with" "critical regulatory molecules"
"Ras" "fails to interact with" "critical effector molecules"
"Ras" "is" "transformation defective"

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

"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.

"Transferase reaction" "is essential to" "the function of Ras"
"Transferase reaction" "is essential to" "the function of at least 20 other farnesyl proteins"

Thus, FTIs are not truly Ras-specific inhibitors.

"FTIs" "are not" "Ras-specific inhibitors"

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

"Severl FTIs" "have been developed as" "potential anti-cancer drugs"

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).

"Potent FTIs of diverse chemical structures" "inhibit" "tumor growth in nude mouse xenograft models"
"Potent FTIs of diverse chemical structures" "inhibit" "tumor growth in a variety of transgenic mouse tumor models"
"Some mouse models" "overexpress" "Ha-ras"
"Some mouse models" "overexpress" "Ki-ras"
"Some mouse models" "overexpress" "N-ras"

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.

"Structurally distinct FTIs" "have" "similar effects"
"Effective doses" "block" "substrate protein farnesylation"
"Structurally distinct FTIs" "achieve" "anti-tumor activity"
"Structurally distinct FTIs" "inhibit" "farnesyl-protein transferase"

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

"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.

"FTIs" "were originally thought to be" "cytostatic agents"
"FTIs" "induce apoptosis in" "tumor cells"
"Aapoptosis" "occurred in" "preclinical tissue culture"
"Aapoptosis" "occurred in" "transgenic tumor models"

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).

"Induction of apoptosis" "occurs by" "caspase-3 activation"
"Induction of apoptosis" "is independent of" "wild-type p53 function"
"p53 loss" "is associated with" "resistance to chemotherapy"

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).

"FTIs clinical trials" "began" "nearly 20 years after the discovery of Ras"
"FTIs clinical trials" "began" "about 9 years after the discovery of Ras farnesylation"

At least 4 different FTIs are currently undergoing evaluation: R115777; SCH66336; L-778,123; and BMS-214662 (24) (Table 1).

"R115777" "is currently undergoing" "evaluation"
"SCH66336" "is currently undergoing" "evaluation"
"L-778,123" "is currently undergoing" "evaluation"
"BMS-214662" "is currently undergoing" "evaluation"

R115777 and SCH 66336 are administered by the oral route, L-778,123 is given by continuous infusion, and BMS-214662 is administered either orally or intravenously.

"R115777" "is administered by" "orally"
"SCH 66336" is administered by" "orally"
"L-778,123" "is given by" "continuous infusion"
"BMS-214662" "is administered" "orally"
"BMS-214662" "is administered" "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.

"R115777" "is in " "advanced trials"
"SCH 66336" "is in " "advanced trials"
"R115777" "has reported" "dose-limiting toxicities"
"SCH 66336" "has reported" "dose-limiting toxicities"
"Toxicities" "involve" "bone marrow"
"Toxicities" "involve" "the gastrointestinal tract"
"FTIs" "can have" "general antiproliferative effects on normal tissues"

The doses achieved in the clinic so far with L-778,123 and SCH 66336 were sufficient to inhibit protein farnesylation in readily obtainable tissues such as white blood cells and cells of the buccal mucosa.

"Clinical doses of L-778,123" "were sufficient to inhibit" "protein farnesylation"
"Clinical doses of SCH 66336" "were sufficient to inhibit" "protein farnesylation"
"Inhibited protein farnesylation" "was obtained in" "white blood cells"
"Inhibited protein farnesylation" "was obtained in" "buccal mucosa cells"

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).

"FTIs" "are anticipated to be used in" "combination with other treatments"
"FTIs" "are anticipated to be used with" "paclitaxel"
"FTIs" "are anticipated to be used with" "vincristine"
"FTIs" "are anticipated to be used with" "cisplatin"
"FTIs" "are anticipated to be used with" "5-fluorouracil"
"FTIs" "are anticipated to be used with" "gemcitabine"
"FTIs" "are anticipated to be used with" "cyclophosphamide"
"FTIs" "are anticipated to be used with" "radiation"

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

"Ras activation" "follows" "a series of protein phosphorylation events within the cell"

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

"The Raf protein kinase" "must directly bind to" "Ras-GTP"

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

"Raf" "phosphorylates" "MAP/Erk kinase (MEK"
"Raf" "activates" "MAP/Erk kinase (MEK)"
"MAP/Erk kinase (MEK)" "phosphorylates" "MAP kinase"
MAP/Erk kinase (MEK)" "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).

"Ras-mediated cellular transformation" "has inspired" "development of protein kinase inhibitors"

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

"ISIS-5132" "is" "an antisense oligonucleotide"
"ISIS-5132" "is directed against" Raf
"ISIS-5132" "is in" "phase II clinical development"

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

"ISIS-5132" " causes" "a dose-dependent reduction of c-Raf mRNA levels"

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

"Pharmacodynamic monitoring" "has been performed" "using peripheral blood mononuclear cells"
"Treated patients" "are" "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.

"ISIS-5132" " "is in" "a phase I trial"
"Median reduction of Raf mRNA" "was" "42% at 48 hours"
"Significant inhibitions" "ere observed up to" "15 days"
"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.

"65 patients" "were" "evaluated"
"4 ovarian cancer patients" "remained stable for" "up to 10 months"
"4 pancreatic cancerpatients" "remained stable for" "up to 10 months"
"4 renal cancer patients" "remained stable for" "up to 10 months"
"4 colon cancer patients" "remained 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).

"Two patients" "showed" "disease progression"
"Disease progression" "coincided with" "the loss of suppression of Raf mRNA levels"

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

"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.

"Hall-Jackson et al. " "characterized" "the biological effects of Raf kinase inhibitor, ZM 336372"
"Hall-Jackson et al. " "characterized" "the biological effects of p38 kinase inhibitor, SB 203580"
"Raf kinase inhibitor, ZM 336372" "weakly inhibits" "Raf kinase activity"
"p38 kinase inhibitor, SB 203580" "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.

"Cells treated with ZM 336372" "exhibit" "a paradoxical increase in Raf activity"
"Cells treated SB 203580" "exhibit a paradoxical increase in Raf activity"
"Raf activity" "is measured" "ex vivo"
"These compounds" "may 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-779,450, which inhibits Raf protein kinase activity in vitro, blocks intracellular signaling by Ki-Ras and Ha-Ras.

"ZM 336372" "does not inhibit" "Ras"
"ZM 336372" "does not inhibit" "Raf-mediated cellular transformation"
"Heimbrook et al." "indicates blocakage of" "intracellular signaling"
"L-779,450" "blocks" "intracellular signaling by Ki-Ras"
"L-779,450" "blocks" "intracellular signaling by Ha-Ras"
"L-779,450" "is" a "triarylimidazole derivative"
"L-779,450 "inhibits" "Raf protein kinase activity in vitro"

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

"Two groups" "have described" "novel MEK inhibitors"

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

"Parke-Davis Pharmaceutical Research" "described" "the first MEK inhibitor, PD098059"
"Parke-Davis Pharmaceutical Research" "identified" "PD-184352"
"PD-184352" "is" "a more potent compound"
"PD-184352" "is" "a moreselective compound" (PD-184352)
"PD-184352" "is from" "a coupled biochemical screen"
"This biochemical screen" "includes" "GST-MEK"
"This biochemical screen" "includes" "MAP kinase"
"This biochemical screen" "includes" "the MAP kinase substrate myelin basic protein"

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).

"DuPont Pharmaceuticals Co." "identified" "U0126"
"U0126" "was identified in" "a cell-based assay"
"This assay" "monitored" "AP-1 response elements"
"U0126" "inhibits" "MEK activity"

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

"PD-184352" "does not compete for" "binding to ATP"
"PD-184352" "does not compete for" "protein substrates"
"U0126" "does not compete for" "binding to ATP" or protein substrates
"U0126" "does not compete for" "protein substrates"
"PD-184352" "functions as" "allosteric inhibitors of MEK"
"U0126" "functions 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.

"PD-184352" "blocks" "MAP kinase phosphorylation in cells"
"U0126" "blocks" "MAP kinase phosphorylation in cells"
"Doses" "abolish" "intracellular MEK activity"
"PD-184352" "inhibits" "anchorage-independent growth of several human tumor cell lines"
"PD-184352" "causes cells to adopt" "a flattened morphology"

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

"PD-184352" "inhibited" "tumor growth in mouse tumor xenograft models"

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.

"This surrogate biochemical endpoint" "correlates with" "biological activity"
"Correlation" "provides strong evidence for" "mechanism-based anti-tumor activity"
" 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).

"Blocking lipid-mediated signaling Activation of growth factor receptors" "is associated with" "changes in phospholipid metabolism"

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

"Phosphorylated residues" "bind" "phospholipase C"
"Phosphorylated residues" "are on" "the intracellular domain of these receptors"
Phospholipase C" "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).

"Diacylglycero" "is" "a breakdown product"
"Diacylglycero" "can activate" "some forms of protein kinase C (PKC)"
"PKC-[alpha]" "is a form of protein kinase C (PKC)"
"PKC-[alpha]" "has been implicated in" "cell proliferative processes"
"PKC-[alpha]" "has been implicated in" "tumorigenesis"

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

"PKC-[alpha] expression" "is elevated in" "some human breast tumors"
"Elevation" "is 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).

"Antisense inhibitors to PKC-[alpha] (ISIS-3521)" "are in" "clinical trials"
"Inhibitors of PKC kinase activity (CGP 41251 and UCN-01)" "are in" "clinical trials"

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

"Kinase inhibitors" "potently inhibit" "PKC activity"
"Kinase inhibitors" "are derivatives of" "staurosporine"
"Kinase inhibitors" "are active in" "mouse tumor xenograft models"

CGP 41251 also inhibits the P-glycoprotein transporter, which mediates the multidrug resistance of many advanced tumors.

"CGP 41251" "inhibits" "the P-glycoprotein transporter"
"The P-glycoprotein transporter" "mediates" "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).

"UCN-01 toxicities" "are" "not remarkable"
"CGP 41251 toxicities" "are" "not remarkable"
"Unremarkable toxcities" "may be related to" "high binding capacity"
"UCN-01" "binds" "plasma proteins"
"CGP 41251" "binds" "plasma proteins"
"Plasma protein binding" "might blunt" "anti-tumor activity"

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

"Antisense compound ISIS-3521" "exhibits" "an acceptable safety profile"

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

"Fatigue" "is" "a side effect"
"Fever" "is" "a side effect"
"Thrombocytopenia" "is" "a side effect"
"Fatigue" "is typical of" "phosphorothioate-based antisense compounds"
"Fever" "is typical of" "phosphorothioate-based antisense compounds"
"Thrombocytopenia" "is typical of" "phosphorothioate-based antisense compounds"

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.

"ISIS-3521" "is being tested with" "carboplatin" "in patients with non-small cell lung cancer"
"ISIS-3521" "is being tested with" " paclitaxel" "in patients with non-small cell lung cancer"
"preliminary data" "indicate" "partial responses"

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

"Ras activation" "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).

"The product of this reaction" "activates" "protein kinase Akt"
"Protein kinase Akt" "is" "a suppressor of apoptosis"

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

"PI 3'-kinase Inhibition" "is predicted to" "inactivate Akt activity"
"Akt activity inhibition" "activates" "apoptotic pathways in tumors"

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

"LY 294002" "potently inhibited" "PI 3'-kinase"

This compound inhibits lipid signaling by growth factor receptors.

"LY 294002" "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

"LY 294002" "is combined with" "an FTI"
"This combination" "induces" "apoptosis in attached tumor cells"
tumor cells" "normally do not respond to" "FTI alone"

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

"Inhibitors" "are efective in" "different steps of the signaling pathways"
"Inhibitors" "may be used in" "combination"