Oncogenes and Tumor Suppressor Genes: Therapeutic Implications’ · 2688 Oncogenes and Tumor...

Vol. 3, 2687-2695, December 1997 Clinical Cancer Research 2687

Oncogenes and Tumor Suppressor Genes: Therapeutic Implications’

Sanford A. Stass2 and A. James MixsonUniversity of Maryland School of Medicine, Baltimore, Maryland

21201

AbstractGenetic instability is a hallmark of cancer. Alterations in

DNA through mutations, deletions, and translocations affect

genes that are fundamental to normal cell growth differentia-

tion and programmed cell death. Here, we discuss these alter-

ations as they relate to oncogenes and tumor suppressor genes.

In addition, we describe the implications the changes in onco-

genes and tumor suppressor genes have on designing new

therapeutic strategies for the treatment of cancer.

Introduction

Cancer is a genetic disease. It is characterized by genetic

instability and long-term uncontrolled growth. Normal cellular

function requires control of growth, differentiation, and pro-

grammed cell death. These are complex processes involving a

cascade of cellular events and proteins encoded by DNA. There

are checks and balances in the normal (wild-type) cell DNA

structure that translate normal cell function. Alterations in the

DNA through mutations, deletions, or translocations result in

aberrations that have been well documented to affect cell

growth, differentiation, and programmed cell death. These dis-

ruptions in DNA (genetic changes) and their resulting abnor-

mally functioning proteins manifest in an autonomous and

clonal proliferation of cells that we recognize as cancer.

We now have vast information regarding these genetic

changes. Unfortunately, merely identifying the defects may not

sufficiently translate into therapeutic efficacy. First, one may

not be able to approach all the abnormalities simultaneously.

Second, there are likely interactive effects that we may not

totally understand or be able to reverse. A corollary to this is that

the identification of an abnormality or abnormalities may deflect

our attention from an approachable end point. Third, even if the

genetic defect has been specifically identified, there may not be

an effective therapeutic strategy to reverse it. A case in point is

sickle cell disease. Thus, as we outline the genetic alterations

that follow, we will discuss how therapeutic intervention might

target these molecular aberrations. However, none of the ther-

apeutic strategies have been proven to be effective long-term.

Mutations in tumor suppressor genes, such as p.53, as in the

Li-Fraumeni syndrome, can be injected with germ-line p.53 muta-

I Presented at “Foundations of Clinical Cancer Research: Perspective

for the 21st Century.” a symposium to honor Emil i Freireich, M.D., onthe occasion of his 70th birthday, March 14-15, 1997, M. D. Anderson

Cancer Center, Houston, TX.2 To whom requests for reprints should be addressed, at University ofMaryland, Department of Pathology and Greenbaum Cancer Center, 22

South Greene Street, Baltimore, MD 21201. Phone: (410) 328-1237;

Fax: (410) 328-1813.

tions or can occur sporadically in tumors. Mutations in oncogenes

are also seen in a variety of tumors. Genes coding for DNA repair

proteins can also be mutated (hereditary non-polyposis colon can-

cer) or be abnormally expressed (i.e., reduced activity of DNA

ligase I in Bloom’s syndrome). A defect in the DNA repair process

could provide the mechanisms that predispose to genetic loss and

mutations. A review of the varied genetic changes, involving on-

cogenes and tumor suppressor genes, seen in cancer suggests that

there is, indeed, an underlying defect that allows critical abnormal-

ities in DNA to persist unrepaired (1).

At the nucleotide level, genetic mutations that are associ-

ated with malignancy can be substitutions, additions. or dde-

tions. A second group of molecular genetic changes comprises

the translocations. In this instance, all or part of a gene recom-

bines with a gene at a different chromosomal site. One of the

most thoroughly studied translocations is the t(9;22) (q34;q I I)

or Philadelphia chromosome, which occurs in 95% of CML.3

The cellular ABL gene on chromosome 9 translocates to chro-

mosome 22, where it combines with the BCR gene in a head-

to-tail configuration, resulting in a hybrid BCR-c-ABL protein

(p210), with tyrosine phosphokinase activity that can transform

hernatopoietic cells (2). A second form of translocation, result-

ing in aberrant expression, is the example of an oncogene

translocated to a site regulated by different transcriptional acti-

vators or enhancers. This is seen in Burkitt’s lymphomas, in

which c-myc is translocated in juxtaposition to the immunoglob-

ulin heavy chain locus t(8;14) (q24;l32), resulting in the dereg-

ulation of c-myc, as a result of its proximity to cis-activating

elements within the IgH locus.

An additional form ofgenetic alteration is the amplification

of the gene itself, which can result in overexpression. These

amplified genes can be present in homogeneous staining regions

in a particular chromosome or present in double minute chro-

mosomes outside the normal chromosomal structure. Examples

of overexpressed oncogenes include neu, amplified in breast and

ovarian carcinoma, and n-myc, in neuroblastoma. In addition,

alterations in gene expression might be due to alterations

in DNA-binding proteins, which up-regulate or do not down-

regulate (i.e., activatedjun,fos, ErbA, ets, myb, ,nyc, and so on),

a mutation of the protein binding that affects binding of regu-

lator proteins or increased mRNA presence due to altered DNA.

RNA, or ribosornal protein structure, which hinders or prevents

degradation. In addition to the genetic defects already discussed,

a loss of expression could occur with gene deletion. We know

that large, chromosomal regions or whole chromosomes may be

lost (i.e., -5 and/or -7 in myelodysplasia, or loss ofRb gene in

retinoblastoma).

Although we know intricate details of these molecular

genetic defects in cancer, this has not translated into directed

3 The abbreviations used are: CML, chronic myelocytic leukemia; G-protein, guanine-binding protein; GAP, GTPase-activating protein;CDK, cyclin-dependent kinase.

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2688 Oncogenes and Tumor Suppressor Genes

therapeutic strategies that result in substantial cures. This is

analogous to sickle cell disease. Clearly, we have known the

genetic defect in sickle cell disease for many years. Unfortu-

nately, this has not translated into a therapeutic approach that

can cure the disease, and long-term effective therapy has, so far,

not been possible. Therefore, we should not assume that further

therapeutic advances will result from additional knowledge of

genetic mechanisms. Recently, Dr. Robert Weinberg said that

“for a number of genetic diseases, knowing the genes might not

help the patient one whit” (3). On the other hand, it would seem

that we have not taken full advantage of our knowledge. We

need to refocus our investigations on developing therapeutic

strategies using our knowledge of molecular genetic changes,

with the resultant effects on cell biology, to target our therapeu-

tic efforts. Here, we will focus on an overview of oncogenes and

tumor suppressor genes and how our knowledge in this area

could be directed to therapeutic interventions.

OncogenesThe homology of viral oncogenes found in tumor-produc-

ing RNA viruses (retroviruses) to cellular oncogenes was estab-

lished in 1976 by H. E. Varmus, M. Bishop, and colleagues with

their work on Rous sarcoma virus and the src gene. It is a

mutation or altered expression of these cellular proto-oncogenes

that is associated with cellular transformation. These proto-

oncogenes are classified according to their cellular function, as

follows.

One group is composed of growth factors and includes

c-sis, responsible for producing platelet-derived growth factor,

and Kst-1/K-fgf, encoding angiogenesis growth factor. Their

involvement in oncogenesis is hypothesized to be a direct mi-

togenic effect (platelet-derived growth factor) or constitutive

growth stimulation through an autocrine mechanism.

Growth factor receptors are a second group of proto-onco-

genes. They include erb-B1, erb-B2, c-neu, met, c-fins, kit, trk,

ret, and sea. The structures of these genes include ligand-

binding, transmembrane, and cytoplasmic catalytic domains,

which, in the majority of the proto-oncogenes, are tyrosine

kinase domains. Oncogenic activation results in a constitutive

activation of the receptor in the absence of ligand.

A third group of proto-oncogenes is signal transducers,

composed of cytoplasmic protein kinases (src, raf- 1 , abi, mos,

pim-], fes, fgr, Ick, and yes). Most of the kinases are tyrosine

kinases that have homology in their catalytic domain; however,

some are serine and threonine kinases. Oncogenic activation

appears to affect the negative regulatory domains, allowing

these enzymes to constitutively phosphorylate their substrates.

A fourth group is composed of the GTP-binding oncogenes

(H-, k-, and N-ras, gip2, and gsp). These are G-proteins that are

responsible for transmitting signals from cell surface ligands,

i.e., growth factors and hormones, and neurotransmitting to

effectors, including adenylate cyclase and phospholipase C.

This transduction, mediated by GTP, to GDP involves binding a

GAP. G-proteins, such as the RAS family of oncogenes, are

activated by point mutations or amplification, resulting in al-

tered GTP-binding or GTPase activity, so that there is extended

stimulation of effector enzymes.

The fifth group of proto-oncogenes is the largest and is

composed of transcriptional regulators, including erbA-l,

erbA-2, ets-l, ets-2,fos,jun, myb, c-myc, L-myc, N-myc, rel, ski,

Hoxil, lyt-JO, lyl-1, Ta!-], E2A, PBX-l, Ttg-l, and rhom-2/

Ttg-2. These genes contain functional domains that mediate

DNA binding and protein-protein interactions. Some of the

genes encode proteins that interact with each other to form

heteroduplexes that control transcription of a target gene. Ex-

pression of these genes is precisely regulated to respond rapidly

to proliferation and differentiation signals. In some instances

(i.e., c-myc), activation is through deregulated expression, pre-

venting the cell from terminating its proliferation phase and

entering its differentiation phase.

bc!-2 is the only known oncogene that is identified as a

regulator of programmed cell death. p53 is also involved in

programmed cell death and is discussed later. The BCL-2 pro-

tein functions on the inner membrane of the mitochondria and

prevents programmed cell death. In lymphoid cells, BCL-2 is

thought to maintain the long lifespan of memory B cells and

plasma cells. BCL-2 is located on chromosome 18 and becomes

activated by deregulated expression, resulting from transloca-

tion into the IgH locus in the t(l4;l8) (q32;q2l) abnormality in

lymphomas. Although increased survival may not result in ma-

lignant transformation, the extended lifespan provides lymphoid

expansion, accumulation, and proto-oncogene activation.

Therapeutic Interventionsras. Activated ras oncogene is the acutely transforming

component of Harvey sarcoma virus 15 . There are three cellular

homologues: H-ras, k-ras, and N-ras. The ras superfamily is

part of the large family of G-proteins and is a component of the

growth-prompting signal transduction pathway (4). Therefore,

activating ras mutations in cancer have been extensively stud-

ied. ras mutation in codons 12, 13, and 61 of H-ras, k-ras, and

N-ras are mutational hot spots in a wide variety of premalignant

and malignant lesions. For example, k-ras is mutated in nearly

90% of pancreatic carcinomas, 50% of adenomas and adeno-

carcinomas of the colon, 30% of adenocarcinomas of the lung,

20-30% of male germ-cell tumors (including N-ras), and 30%

of rnyelodysplasias and acute myeloid leukemias (including

N-ras), and 60% of CMLs (including N-ras). This diverse

involvement of the ras mutation in cancer makes it a likely

target for therapeutic intervention.

Several therapeutic approaches are possible. These ap-

proaches need not necessarily be limited to ras because similar

approaches could be used in other activated genes (mutated

genes) to block transformation.

The approach of ras antisense takes advantage of a tech-

nique that has been used in basic molecular biology to study the

effects of blocking expressed genes at the mRNA level. The

principle is based on the ability of complementary nucleic acids

to bind to each other. The mRNA transcribed from the gene

encoded in DNA is oriented as sense in mRNA. Therefore, the

antisense molecules, which are constructed to be complemen-

tary to a specific sense mRNA, bind to it and prevent its

processing and translocation. An alternative action is thought to

be mediated by RNase H, an endonuclease that cleaves the RNA

nucleotides of RNA-DNA duplexes. Antisense therapy is de-

signed to be specific for the target. Because we know the



Clinical Cancer Research 2689

molecular genetic abnormality or abnormalities in many tumors,

cancer is an excellent model for its use. Furthermore, ras mu-

tation is an excellent target because of its involvement in so

many tumors.

However, there are many disadvantages. First, one has to

assume that targeting a single molecular genetic event is suffi-

cient to totally inhibit malignant proliferation. Cancer is a corn-

plex process, and so one can not assume that, if mutated ras

mRNA and its protein were totally inhibited, the malignant cell

would be permanently inhibited. Certainly, evidence indicates

that close to 100% of rnRNA can be inhibited, with resultant

inhibition of growth. However, cells have alternate mechanisms

for growth, and a bypass process is possible. depending on the

“total” initiating factors. Thus, the inhibition of ras mRNA may

ultimately prove to be ineffective. One form of antisense used is

the oligonucleotide. Oligonucleotides are usually designed as

small stretches of DNA, from 8 to 40 or 50 nucleotides long,

that inhibit pre-mRNA and mRNA. The protein translation start

site, exon-intron borders, and the region adjacent to the mRNA

cap site appear to be the most effective sites to target, suggesting

an inhibition of RNA interaction with cell function that is

necessary for translocation of mRNA into protein. Unfortu-

nately, antisense oligonucleotides are degraded rapidly by en-

donucleases, and stability needs to be improved for effective

use. Furthermore, the affinity and permeability of antisense

oligonucleotides represent potential obstacles. As a result, large

and repeated doses of oligonucleotides are necessary. However,

this does not necessarily result in delivery of the oligonucleo-

tides to all the tumor cells. Recently, improved oligonucleotides,

other than phosphorthioates, have been developed to circumvent

degradation and delivery difficulties.

The introduction of sequences that can be transcribed to

produce an antisense molecule inside cells is an alternate ap-

proach. The advantage of this antisense RNA approach is the

continuous generation of antisense sequences in the cell as a

result of stable transduction, using vectors (such as retroviruses)

expressing antisense RNA. The disadvantages are inefficient

transduction into the cell, lack of an ideal vector that is relevant

to the target cell, resulting in 100% of the cells being trans-

duced, and failure to produce long-term expression of the trans-

duced antisense due to a number of unknown factors, which are

currently under investigation. Retroviral vectors, such as Mob-

ney leukemia virus backbone, are commonly used because they

transduce dividing cells. However, high viral titers are needed

because it may be that as few as 1 in 100 viruses infect the cell.

Furthermore, there is essentially no information on the effects of

retroviral transduction on the target cell. Insertional mutagene-

sis, induction of other genes, and effects on immune response

are only a few of possible effects. We are currently studying

these potential effects in our laboratory.

Retrovirus-mediated introduction of antisense RNA has

been studied in a human lung cancer model using K-ras. Using

H460 cells (human lung adenocarcinoma with a point rnutation

at codon Gl of K-ras), a 95% decrease in K-ras production was

seen. The H460 cells expressing the antisense in culture showed

a 3-fold decrease in cell growth and did not produce tumors in

nude mice. Using a retroviral vector (instead of electroporation),

we targeted H460 cells after intratracheal injection in nude mice.

Only 10-14% of mice treated developed tumors, in contrast to

73-100% of controls (5). A Phase I trial is now underway.

Using antisense ribozymes is another potential approach.

Ribozymes are RNA molecules that are capable of catalyzing

nucleic acid hydrolysis at specific residues. There are groups of

self-cleaving ribozymes, called “hammerhead” and hairpin, that

have a consensus secondary structure. Both the hammerhead

and hairpin have catalytic motifs that can be incorporated into

an antisense RNA, creating an enzymatic antisense RNA. The

antisense portion of this hybrid ribozyrne gives target specificity

and the ability to cleave multiple substrates derived from the

catalytic component. In vitro studies have shown some success

with ribozymes. Ribozymes have been shown to target H-ras

and N-ras RNA substrate but not a wild-type RNA substrate.

This has also resulted in altered growth. In a study in nude mice,

mice with bladder carcinoma administered intravescilarly lived

longer when ribozyme was transfected into their tumors. How-

ever, the use of ribozymes has similar problems to the use of

oligonucleotides, including delivery to the target, stability

within the cell, and a high turnover rate (6, 7).

Several additional approaches to inhibiting the effects of

ras are possible. One approach would be to target amino acids

32-40 of p2l’�. This is the binding effector domain that is

involved in binding GAP, which results in a cascade of signals

downstream. Potentially, the injection of an inactive form of ras

with a mutation at this site competes for downstream effectors.

Obviously, this strategy has the same difficulties as mentioned

above.

One therapeutic approach that is under active investigation

by major pharmaceutical companies is inhibition at several

potential sites of the cholesterol biosynthetic pathway. It has

been shown that both the normal and mutated ras oncogene

must be attached to the inner surface of the plasma membrane to

function. This occurs through a farnesyl group attaching to the

cysteine of the COOH-terminal sequence CAAX (A represents

any aliphatic acid, and X is any residue) by farnesyl transferase.

Following attachment of the farnesyl group, -AAX is cleaved.

This is a common mechanism for proteins to attach to mem-

branes.

Inhibition of the farnesylation of ras will inhibit function.

However, this is not a selective approach because both wild-type

and mutated ras will be inhibited. Posttranslational modification

of ras, because it is not preferential for mutated ras, could block

other essential proteins. Still, this may be an attractive approach

because cellular dependence on oncogenic ras might result in

tumor cells being more sensitive than normal cells to the action

of a farnesyl transferase inhibition. Inhibitors of CAAX farnesyl

transferase have been identified, including compounds from

Streptomyces related to a-hydroxy-farnesyl, tetrapeptides, and

their analogues, such as L-73l,734, developed by Merck, and

benzodiazepine peptidomimetics (8). Another approach is the

use of inhibitors of the production of farnesyl diphosphate,

which is a substitute for farnesyl transferase and is synthesized

in the cholesterol biosynthetic path from mevalonic acid. It is

hypothesized that the interference with farnesyl production will

translate into decreased ras activity. A drug used to lower

cholesterol, such as lovastatin, inhibits hydroxymethyl glutaryl

coenzyrne A reductase, which is the initial and rate-limiting step

of cholesterol biosynthesis. Lovastatin, in vitro, has been shown




4 S. A. Stass, E. J. Freireich, and C. Platsoucas, unpublished data.

to inhibit NIH3T3 cells that are transformed by H-ras and

human pancreatic carcinoma cells in nude mice. However, be-

cause many proteins are affected by cholesterol biosynthesis, it

is difficult to assign the effect to ras. Unfortunately, 10 times the

cholesterol-lowering dose is necessary to suppress tumors (9).

One other potential therapeutic approach would be immu-

nization with mutated ras protein. These mutations produce

novel proteins, which should be seen as immunogenic by the

host. There is preliminary evidence in vitro and in mice that

mutated ras peptides can generate a T-cell response. However,

this response may be nonspecific. We have performed similar in

vitro studies using bcr-abl and have found both nonspecific and

rarely specific T-cell cytoxicity.4

One other issue that requires consideration is the associa-

tion of ras mutations with resistance. The ras mutation has been

associated with poor prognosis. However, there are little data to

indicate that ras mutations are associated with chemotherapeutic

resistance. One exception is that there are in vitro data to suggest

increased resistance to all-trans-retinoic acid. There is also

evidence to suggest that ras mutations are associated with

radiation resistance, which could be reversible by use of ras

inhibitors, as discussed above.

The HER-2/neu Gene

This gene represents an oncogene that has been found to be

amplified or overexpressed in a wide variety of human primary

tumors, especially adenocarcinomas (10). This gene was origi-

nally isolated as an amplified V-erb-related sequence in human

breast cancer, salivary gland adenocarcinoma, and gastric can-

cer. It has been most frequently studied in breast cancer. The

HER-2/neu gene has been found to be amplified or rearranged in

breast cancer. There is a 2-20-fold amplification in about 30%

of primary human breast cancer. There is a significant correla-

tion between level of HER-2/neu gene amplification and time to

relapse and overall survival (1 1). HER-2/neu can be coamplified

with other proto-oncogenes, i.e., erbA. Other tumors reported to

have HER-2/neu amplification include ovarian carcinoma, gas-

tric carcinoma, colon cancer, lung cancer, and cervical cancer,

further supporting the role of HER-2/neu in human carcinogen-

esis.

In terms of therapeutic implications, it seems that breast

tumor with HER-2/neu overexpression may be less responsive

to adjuvant therapy. Non-small cell lung cancer HER -2/neu-

overexpressing tumors may have intrinsic chemoresistance. This

is an area for investigation. Targeting p1 85 neu on the surface of

HER-2/neu-overexpressing tumor cells is an attractive therapeu-

tic strategy. One approach would be the use of monoclonal

antibodies that bind to the extracellular domain of the p185

protein, which has been shown to inhibit tumor formation and

tumor metastasis in animal models (12). This approach may also

increase chemosensitivity to drugs such as cisplatin and Taxoi.

Gene therapy also represents a possible approach. The adeno-

virus EJA gene product has been identified as a viral suppres-

sion gene for ras-induced metastasis, and it has been demon-

strated the E1A represses neu expression via a CIA DNA

element in the neu promoter (13). It was shown that introduction

of the EJA gene into neu-transformed 3T3 cells reduced the

formation of experimental metastatic tumors (14). However,

EJA may also suppress transformation features of human cancer

that do not overexpress HER-2/neu. Animal studies have shown

that liposome-mediated EJA gene therapy may serve as a pow-

erful therapeutic reagent for HER-2/neu-overexpressing human

ovarian cancers by targeting the overexpressed HER-2/neu on-

cogene (15). This approach has also been used to produce a

liposomal-p53 complex to suppress primary and metastatic hu-

man breast tumors in a nude mouse model (see below).

Chromosomal TransbocationsAs previously mentioned, chromosomal transiocations,

many of which have been especially well characterized in leu-

kemias, result in translocated DNA regions, which produce

hybrid messages. This may play a role by in transformation by

affecting cell growth and/or differentiation. Many translocations

have been identified and characterized. These translocations and

the genes involved are reviewed in several publications (for

review see Ref. 16). Some common translocations and/or trans-

locations of particular biologic interest with affected genes are

listed in Table 1. Clearly, the therapeutic approach to translo-

cations is potentially similar to that discussed with ras. This

approach includes antisense, ribozymes, and retroviral con-

structs to inhibit fusion transcripts. Unfortunately, one cannot be

sure that, in the hybrid, transcripts are fully responsible for

ieukemogenesis. Therefore, blocking them may not result in

inhibition of leukemic growth. Furthermore, the translocation

abnormality affects cellular development and other complex

cellular functions (such as transcription) that are not easily

reversible. Finally, even if blocking the transcript was therapeu-

tically effective, doing so in all cells is virtually impossible

unless the appropriate hematopoietic stem cell (committed ear-

tier) is targeted. This approach might be feasible for bone

marrow purging through organs, although targeting all abnormal

cells is a major obstacle. Currently, in our laboratories, we are

focusing on identifying the appropriate hematopoietic stem cell

and delivery system for genetic modification that will result in

long-term hematopoietic reconstitution (in the transplant set-

ting) and long-term gene expression.

Other approaches to targeting hybrid transcripts could be

considered. With CML as a model, the use of tyrosine kinase

inhibitors might be of value. There are several candidate tyro-

sine kinase inhibitors, including tyrphostin, herbimycin A, erb-

statin, and geristein (17). However, because tyrosine kinase is

important in growth and development of normal cells, these

inhibitors would affect normal tissue and, thus, have the poten-

tial for significant toxicity.

Tumor Suppressor GenesThe tumor suppressor genes that have been most widely

studied are the retinobiastoma gene (Rb) and p53. We will focus

on these two tumor suppressor genes as models for therapeutic

intervention. Other tumor suppressor or potential tumor suppres-

sor genes have been reviewed, including the Wilm’ s tumor gene

(WTI), the APC and DCC genes in familial adenomatous pol-



Cytogenetic rearrangements Disease Genes involved/functions

MYC-nuclear HLH proteinBurkitt’s lymphoma,ALL-L3 (B-ALL)

Pre B-ALLT-ALL

T-ALL

T-ALL

T-ALL

T-ALLT-ALL

t(7;i9) (q35;pl3)

t(8;14) (q24;qll)

(8;l2) (q24;q22)

del(i) (p32)

T-ALLT-ALLB-CLL

T-ALL

CML, B-ALL, AML

Pre-B-ALL

APL

AML-M2

AMLAML-M2

AML-A4

AML-M4Eo

AML-M4AML-M5

AML

a CLL, chronic lymphocytic leukemia; ALL, acute lymphocytic leukemia; AML, acute myeloid leukemia; HLH, helix-loop-helix; APL, acuteprornyelocytic leukemia.


Table 1 Molecular characteristics of neoplastic rearrangements in acute leukemia”

Type of oncogene activation

Juxtaposition to immunoglobulin

regulatory element

Juxtaposition to T-cell receptorregulatory element

Juxtaposition to other thanimmunoglobuliniT-cell

receptor loci regulatoryelement

Fusion genes

t(8;14) (q24;q32); t(2;8) (pl l;q24);

t(8;22) (q24;ql 1) breakpoints arescattered over 350 kbp

t(5;i4) (q31;q32)

t(ii;14) (pi5;qll)

t(1l;l4) (pi3;qll) breakpoints areclustered within 2 kbp; variant

translocation t(7;i 1) (q35;pi3)t(lO;i4) (q24;qi 1) breakpoints are

clustered; variant translocations

t(7;iO) (q34;q24)

t(l;14) (p32;qll) clustered in 3’

untranslated region or S’

untranslated region; varianttranslocation t(l ;7) (p32;q34)

t(7;9) (q34;q34)

t( 1;7) (p34;q34)

t(7;9) (q34;q34.3) T-ALL

t(9;22) (q34;qi 1); most of them areclustered

t(i;l9) (q23;pi3); most of them areclustered

t(i5;17) (q22;ql 1-12); clusteredbreakpoints

t(8;2i) (q22;q22); clusteredbreakpoints

t(6;9) (p23;q34); clustered breakpoints

inv(l6) (pl3.l;q22)

t(l6;16) (p13.1;q22)

t(9; 1 1) (q22;q23)t(6;l 1) (q27;q23)

t(li;i9) (q23;pi3)

5q-

Interleukin-3 growth factorTtg-1/rhombotin-l Lirn domain

protein a, transcription factor

Ttg-2/rhombotin-i Lim domain

protein a, transcription factor

TCL3/HOXJI, a homeobox gene

TCLS/SCL/TAL1

TAL2-HLH protein

LCK-p56�, member of Src family;tyrosine kinase

TAN-human homologue ofDrosophila Notch gene

LYLJI-HLH protein

MYC-HLH protein

BTG-1 deregulated MYCSIL deregulates TAL-l; TAL-1-HLH

protein SIL, nuclear protein withsome homology to topoisornerase 1

BCR-ABL BCR, GAP for p2l� ABL,tyrosine kinase

E2A-PBX; E2A is an HLH; PBX is ahomeodomain protein

PML-RARA and RARA-PML, a zincfinger protein; RARA-Zn, finger-

retinoic acid receptor, fusionproteins

Transcription factors; AMLJ-ETOfusion protein

DEK-CANDEK nuclear

CAN cytoplasmic; transcriptionfactors; DEK-CAN, SET-CANfusion proteins

MYHLUCBFB, transcription factor

MU (ALU), homeobox regulator,trithorax homology, fusion proteins

Unknown

yposis, VHL gene in Von-Hippel-Lindau, and NFl and NF2

genes in neurofibromatosis (1).

The tumor suppressor genes represent the antipathy of

oncogenes. They normally function to suppress and regulate cell

growth. Usually, a loss of both alleles of a tumor suppressor

gene is necessary for failure to suppress and control cell growth.

Mutations that occur in one allele are recessive and can be

transmitted to the next generation. Individuals with a mutation

in one allele are obviously at a greater risk to develop a malig-

nancy because the molecular genetic abnormality can adversely

affect the normal tumor suppressor gene allele resulting in

transformation. In Rb, loss of normal function of one Rb gene

appears to be adequate to result in malignant transformation.

This is not always the case because, in some instances, other

events may be necessary for malignant transformation. The

report that fusion of nontumorigenic human cells with tumori-

genie human cells resulted in hybrid cells that could no longer

form tumors supported the concept that there was genetic infor-

mation in normal cells that could suppress cancer cells. This

would indicate that malignancy could be caused by abnormali-

ties of genes (mutation or deletion) capable of controlling and

suppressing cell growth ( 1 8). Karyotypic analysis (approximate-

ly 5% of Rb tumors have a i3qi4 deletion), restriction fragment

length polymorphism analysis in tumors and families, examina-




tion of variable numbers of tandem repeats in paternal and

maternal alleles, and sophisticated chromosome transfer tech-

niques (microcells), in which smaller chrornosomal portions are

transferred into tumor cells, resulting in inhibition of tumor

growth, have enabled the chromosomal localization and, ulti-

mately, identification of tumor suppressor genes.

The retinoblastoma gene Rb! was cloned using i3ql4-

specific probes and was later further characterized (19, 20). The

product of the Rbl gene is a 928-amino acid protein, which is

phosphorylated at multiple sites with three functional domains

(oligimerization, pocket, and DNA-binding/transcription fac-

tor). The pocket domain can bind to viral oncoproteins. The Rb

protein is phosphorylated at several sites by cdc2 kinase. Phos-

phorylation of Rb is critical to its activity. and its biologic

activity is regulated by phosphorylation. Inactivation of Rb!

leads to tumor formation in various cell types. Thus, Rb protein

is involved in controlling cell growth. Tumor cells lacking Rb

can demonstrate control of growth by introducing normal Rb!

alleles. Furthermore, phosphorylation of Rb is essential to cell

growth suppression. The data that show that viral oncoproteins

(large T antigen of SV4O, E1A protein of human adenovirus

type 5, E7 protein ofhuman papillomavirus types 16 and 18, and

EBNA-5 protein of EBV) bind to hypophosphorylated Rb and

that Rb is hypophosphorylated through early G1 , with hyper-

phosphorylation in 5, G,, and M. suggest that the hypophos-

phorylated form of Rb has activity that suppresses cell division,

which can be reversed in late G1 by phosphorylation or onco-

protein binding. Purified unphosphorylated Rb injected into

cells results in G1 arrest, further supporting this concept. Fur-

thermore, the data support that cyclin-cdk complexes are re-

sponsible for hyperphosphorylation of Rb, with resultant release

of the cell from growth control. In addition, binding of onco-

proteins to Rb protein can inactivate Rb function, which results

in loss of controlled cell growth and malignant transformation.

Rb can bind E2F, a cellular transcription factor. E2F binds to the

adenovirus EZ promoter and activates E2 expression and also

binds to genes activated in mid to late G1 (c-mvc, cdc2 dihy-

drofolate reductase, and other genes involved in DNA synthe-

sis). It appears that Rb can block the ability of E2F to activate

transcription of the aforementioned genes and that E2F is im-

portant in initiating signals for the cell to enter S phase (21).

This data suggests that the ability of Rb to suppress activation of

E2F-mediated transcription is essential to the growth-suppress-

ing activity of Rb and that viral oncoproteins disrupt the inter-

actions between Rb and E2F, which may explain the ability of

viral oncoproteins to transform (22). Rb is also involved in

interaction with genes involved in cell cycle control to an extent

that Rb dysfunction could disrupt the normal cell cycle.

Future therapeutic interventions using tumor suppressor

genes, such as Rb, are possible. One approach is gene therapy

using delivery systems for the Rb gene, such as retroviral,

adenovirus-associated, or liposomal systems. The limitations are

the same as those previously mentioned, including targeting a

sufficient cell population to have an effect and inherent prob-

lems with delivery systems (insertional mutagenesis, immune

response. transduction efficiency, and so on). However, we

anticipate that these obstacles will be overcome by more effi-

cient carriers. These obstacles could be overcome especially if

there was a “bystander” effect, as has been seen with liposomal-

p53 complexes (see below). One additional approach might be

to use Rb protein as a therapeutic agent. There are indications,

in vitro in leukemia, that Rb protein may have a suppressive

effect whether or not there is a mutation in Rb (23). Such an

approach might bypass the difficulties with gene therapy. This

approach might also be effective for other tumor suppressor

genes, such as p.53.

In conclusion, our knowledge regarding the molecular ge-

netics of oncogenes and tumor suppressor genes in cancer is

now vast. The challenge is to now effectively translate this

information into therapeutic strategies that result in short-term

and long-term benefits to the patient.

p5.3. In 1979, Arnold Levine of Princeton University and

David Lane of the University of Dundee independently discov-

ered the p53 protein. However, the p53 protein initially ap-

peared to act only as an oncogene and not as a tumor suppressor.

This was due to scientists inadvertently working with a mutant

form of p53 gene. It was not until 1989 that Levine and

Vogelstein discovered that p.53 did, indeed, suppress cancer.

Approximately, 60% of tumors are known to have a p53 rnuta-

tion, including those of breast, lung, liver, skin, prostate, blad-

der, cervix, and colon. More than 90% of p53 mutations are

localized between codons 120 and 290 in the DNA-binding

domain of the p53 protein (24). Furthermore, the vast majority

of p53 mutations are missense mutations (85.6%). Although

mutations of the p.53 gene are most frequently acquired, they can

also be inherited through the Li-Fraumani syndrome. In these

families, one mutant p53 allele is inherited, and the second allele

acquires a mutation. In this syndrome, members of these fami-

lies frequently developed breast cancer, sarcomas, and colon

and lung cancer. Similarly, mice harboring a p.53 allele have a

higher incidence of cancer than do normal mice with wild-type

p53 genes. p53 is not only important because it mutates fre-

quently in a number of malignancies but also because it plays an

increasingly important role in the diagnosis. The presence of

mutations in the tumor suppressor genes, particularly p.53, have

been associated with increased tumorigenicity, metastases, and

mortality in human breast cancer (25, 26) and many other

cancers. We will first focus on the function ofp53 and using p53

for the therapy of malignancies.

It is now known that p53 plays a critical role in the cell

cycle. p53 coordinates multiple responses to DNA damage.

DNA damage results in an increase in the level of the p53

protein. Following DNA damage, an important function of

wild-type p53 function is to control the progression of cells from

I to S phase. Recently, several groups have found that p53

transcriptionally activated a p21 kd protein (also known as

WAF1 , Cipi , Sdii, p2OCAP, and Picl), an inhibitor of CDKs

(27, 28). Inhibition of CDK activity is thought to block the

release of the transcription factor E2F and related transcription

factors from the retinoblastoma protein Rb, with consequent

failure to activate transcription of genes required for S-phase

entry (28, 29). Evidence that is consistent with the model that

pRb is a downstream effector of p53-induced G1 arrest has

recently been reported (30).

In addition to cell cycle arrest, p53 also induces apoptosis.

The molecular details of this form of cell death are not fully

understood, but conserved features include activation of pro-

teases of the ICE/Ced-3 class and regulation by members of the




Bcl-2/Ced-9 family (31, 32). p53 has been shown to induce

expression of BAX, a gene encoding a positive regulator of this

pathway (33). A second way in which p53 may induce apoptosis

is through the repression of transcription. This function is me-

diated via TATA box elements when sites for p53 binding are

absent in the promoter of the target gene (28, 29). For example,

Bcl-2 can be repressed, perhaps by p53 binding to the TATA-

binding protein and inhibiting its function as a basal transcrip-

tion factor.

Depending on the cell type, an increase in p53 can cause G1

arrest and/or apoptosis. Apoptosis is promoted by some of the

Bcl-2 homologues (Bax, Bak, Bad, and BclXJ. Other members

of the Bcl-2 family (Bcl-2, BcXL, and Bag- 1 ) inhibit apoptosis

by forming dimers with Bcl-2 homobogues that promote apop-

tosis (34). The various ratios of the Bcl-2 family members may

result in either suppressed or accelerated apoptosis in response

to accelerated stimuli. These two cellular outcomes, G1 arrest

and apoptosis, are also influenced by growth factors, oncogenes,

and activation of cytokine-like signal transduction pathways

(35).

It has also been well established that genes induced by p53

include: MDM2, the product of which is a negative regulator of

p53 (36); thrombospondin I, which inhibits angiogenesis (37);

IGF-BP3, which may be an autocrine/growth regulator through

its interaction with IGFJ (38); cyclin G, the function of which

has yet to be determined (36); and GADD45, which may play a

role in DNA repair (36, 39). Besides inducing GADD4S, there

is also some evidence that p53 may play a role in DNA mis-

match repair by directly binding to the DNA (40, 41). Of the

proteins that p53 is known to induce, only thrombospondin I and

IGF-BP3 are secreted from the cell.

Because p53 is a central regulator in controlling the cell

cycle, one might expect that restoration of wild-type p53 func-

tion can inactivate the proliferative effects of the mutated prod-

uct. In fact, various groups have found that in vitro transfection

of a tumor suppressor transgene into a variety of tumor cells

decreased the tumor growth rate in ex vivo experiments (42-47).

One might expect that restoration of the wild-type p.53 gene

into tumors is technically difficult. Although efficient transfer

into tumors is rarely possible, various groups have reported that

p53 decreases tumor growth by a bystander effect and not

necessarily by efficient restoration of p53 into the tumor. In an

orthoptic lung cancer model, Roth’s group reported that retro-

virally transfected p53 inhibits developing lung cancer in mice,

despite the fact that p53 had only been transfected into 30% of

the tumor cells (46). These studies have been extended to

clinical studies involving lung cancer patients in a terminal state.

Of the nine patients with lung tumors treated with p53, three had

tumors that did not increase in size, and three had their tumors

decrease in size. In fact, six separate biopsies of one tumor after

retroviral injection showed no evidence of a viable tumor.

Ionizing radiation or DNA-damaging agents, such as cis-

platin, when coupled to p53, have been found to be synergistic

in their antitumor activity (48). Direct injection of a p53 ade-

novirus construct into lung tumors, followed by i.p. administra-

tion of cisplatin, induced massive apoptotic destruction of the

tumors (49). Currently, there are three gene therapy p53 clinical

trials in Phase I using viral vectors.

Another novel gene therapy approach is the use of a EIB

55-kb gene attenuated adenovirus. This virus selectively repli-

cates in and causes the lysis of p53-defective tumor cells but not

cells with functional p53 (50). Replication in and cytolysis of

normal cells are reduced 100-1000-fold in vitro and in vivo by

this gene deletion. Injection of this mutant virus into p53-

defective human cervical carcinomas grown in nude mice

caused a significant reduction in tumor size and caused corn-

plete regression of 60% of the tumors. Thus, this promising

result has clinical implications, but its use for clinical trials is

limited to local therapy at present.

The vast majority of ex vivo and in vivo experiments have

used viral vectors that are known to have a high transfection

efficiency to restore p53 in the p53-deficient tumor cells. How-

ever, toxicity of these viral vectors precludes them from being

given systemically to treat metastatic disease, whereas it has

been demonstrated that liposome-DNA complexes are not toxic

(Si).

Although transfection efficiency of liposomes complexed

to DNA is not thought to be highly efficient (52), we demon-

strated that systemic administration of a liposorne-p53 complex

significantly affects tumor growth and metastases of breast

cancer cells injected into nude mice (53). Surprisingly, the

transfection efficiency of the tumor by the liposome-p53 corn-

plex was quite low. The mechanism by which systemic admin-

istration of liposome-p53 complex inhibits tumor growth ap-

pears to be a bystander effect (46, 53, 54). Potential mechanisms

for this bystander effect are the following: inhibition of angio-

genesis (37); stimulation of the IGF-BP3 protein (38); and

activation of a immunological response. Although the rnecha-

nism of p53’s bystander effect is unclear, the most important

aspect of this study is that it demonstrates that iv. p53 can

potentially decrease metastatic tumors.

Another approach to the treatment of malignancies is the

potential of using mutant p53 for vaccinations (55). CTLs rec-

ognize processed peptide fragments of any endogenous protein,

after these peptides are carried to the cell surface by MHC class

I molecules. Thus, a tumor antigen does not have to be cx-

pressed as an intact protein on the cell surface to be recognizable

by CTLs. Recently, CD8 + CTLs were induced to specifically

target mutant p53 products. Immunizing BALB/c mice with

spleen cells pulsed with a mutant p53 peptide completely inhib-

ited the development of lung cancer with the same mutation. In

contrast, the wild-type peptide, without the mutation, had no

effect on the growth of the lung tumor. Vaccinations with other

mutant p53 peptides have been found to be effective against

tumors expressing a variety of mutant p53 proteins (56). These

findings point the way toward an approach to selective immu-

notherapy against tumors. Alternatively, canary pox virus vec-

tors expressing p53 as a cancer have been used (49). The

protective effect of this live virus vaccine was not dependent on

the p53 mutation, and wild-type and mutant p53 were both

effective in reducing tumor growth.

When malignancies (lung, breast, colon, prostate, bladder,

or skin) harboring a p53 mutation arise, they tend to be more

aggressive, resist chemotherapy, and respond more poorly to

most protocols than do cancers of similar tissues with wild-type

p53. An exciting area of research is to define novel agents

and/or stratagems (57) that preferentially target p53-defective

cancer cells for destruction. In one study, more than 60,000




34. Boise, L. H., Gonzalez, G. M., Postema, C. E., Ding, L., Lindsten,

T., Turka, L. A., Mao, X., Nunex, G., and Thompson, C. B. bcl-x, a

compounds are being screened for activity against a panel of 60

different cancer cell lines. The cell lines have been characterized

with respect to a number of indices of p53 status, including p53

function and p53 protein expression. Most standard clinical

agents are more active in p53 wild-type cells than they are in

p53 mutants in this particular assay. However, several com-

pounds have now been identified that are more active in p53-

defective cells.

Therapies directed toward proteins that bind to p53 and

inactivate its functions are being developed. MDM2, overex-

pressed in sarcomas and in some breast cancers, is a paradigm

of a cellular protein that binds to the NH,-terminal region of the

p53 protein. Antisense oligonucleotide to decrease the levels of

the MDM2 protein or drugs (i.e., peptide) that directly interfere

the binding of MDM2 to p53 are being advanced (58). Simi-

larly, therapies are being developed to prevent the formation of

the p53-E6 protein complex. E6 is a protein form of the onco-

genie papilbomavirus, which has been associated with cervical

and rectal carcinomas.

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Oncogenes and Tumor Suppressor Genes: Therapeutic Implications’ · 2688 Oncogenes and Tumor...

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