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(CANCER RESEARCH 49, 3713-3721, July IS, 1989]
Perspectivesin CancerResearch
Oncogenes, Antioncogenes, and the Molecular Bases of Multistep Carcinogenesis1
Robert A. Weinberg2
Whitehead Institute for BiomédicalResearch and Massachusetts Institute of Technology, Cambridge, Massachusetts 02142
Tumorigenesis in humans and laboratory animals is a com
plex, mu 11¡stepprocess (1, 2). In humans, in whom the process
has been studied only indirectly, measurements of age-depen
dent tumor incidence indicate kinetics dependent on the fifth
or sixth power of elapsed time (3). This suggests a succession
of five or six independent steps, each of which is rate limiting
on the process. In experimental models, such as mouse skin
tumorigenesis, the process has been broken down into at least
three distinct steps: initiation, promotion, and progression (4,
5). From the perspective of the organism, the multistep nature
of tumorigenesis is easily rationalized; each step in the process
represents a physiological barrier that must be breached in
order for a cell to progress further toward the end point of
malignancy. Such multiple barriers conspire to ensure that
successful completion of the tumorigenic process is a rarely
achieved event.
An unanswered question concerns the natures of these bar
riers to tumor inception and growth. A portion of the defenses
may well derive from systemic defenses against tumors; yet
others, confronted in this essay, reflect underlying mechanisms
governing the behavior of individual cells. Our cells and likely
those of all metazoa would seem to be constructed so as to
present multiple impediments to full malignant transformation.
Only recently has it been possible to search for the molecular
and cellular mechanisms that govern multistep tumorigenesis.
What are the rules that govern cell growth? How is the growthregulatory circuitry laid out within the cell? And how can
multiple physiological controls be overridden to produce the
deregulation of neoplasia?
Collaborating Oncogenes
The oncogene paradigm developed over the past decade has
proved to be particularly powerful in generating an explanation
of cancer at the molecular level. Because cellular oncogenes are
mutated forms of normal cellular genes, they provide clear
indication of the genetic targets that suffer alteration at the
hands of mutagenic carcinogens. Accordingly, a simple model
would identify protooncogenes, the normal antecedents of cel
lular oncogenes, with the genetic targets the alteration of which
defines each of the distinct steps in multistep cellular transfor
mation. In this view, the evolutionary history of a tumor cell
clone is demarcated by a series of oncogene activations, each of
which confers on the tumor cells some of the phenotypes that
in aggregate constitute fully malignant behavior.
An initial connection between oncogenes and the multistep
nature of tumorigenesis was made 7 years ago through studies
of two viral oncogenes, the middle T (A/7") and large T (LT)
genes of polyomavirus (6). Neither was found to be able to
transform rat embryo fibroblasts on its own. However, the two,
working in collaboration, elicited a fully tumorigenic phenotype. This suggested that each oncogene was specialized to
Received 2/2/89; accepted 4/19/89.
1Part of the work described herein was supported by National Cancer Institute
Grant OIG 5 R35 CA39826.
2American Cancer Society Research Professor.
induce part of the phenotypes required for full transformation.
Was this a peculiarity of these viral oncogenes or were the
principles transferable as well to the behavior of oncogenes
derived from the cell genome?
The model was indeed extended to a number of oncogenes of
cellular origin. Thus, neither a ras nor a myc oncogene was
found able to induce full transformation while the two, cointroduced into rat embryo fibroblasts, achieved this end result
(7). Analogously, a ras oncogene was found to collaborate with
the adenovirus EIA oncogene in the full transformation of baby
rat kidney cells (8).
These rather simple experiments had a number of conceptual
ramifications that warrant mention. Like the polyomavirus
genes, the observed ability of ras and myc oncogenes to collab
orate with one another in the transformation process showed
that each acts in a distinct, complementary way on cell phenotype. Detailed studies in rodent cells showed that rus oncoproteins can induce refractility, anchorage independence, and
growth factor secretion even when expressed in low amounts;
such expression did not favor immortalization in culture. Con
versely, the myc oncoproteins appeared more adept at immor
talization and less able to induce anchorage independence and
growth factor secretion (9, 10).
Such distinctions in function have been seen in a variety of
cell backgrounds. They suggest that the cell is organized so as
to respond in only limited ways to the transforming influences
of a single activated oncogene. Stated differently, they suggest
that an activated oncogene is able to control only a limited
subset of the growth-regulatory circuits of the cell. These cir
cumscribed actions of single oncogenes presumably reflect the
correspondingly limited powers of antecedent protooncogenes,
each of which has apparently been evolved to transduce only
part of the complex information regulating cell growth and
quiescence.
Yet other oncogenes could be placed into two functional
categories based on their abilities to complement either a ras
or a myc oncogene in transformation assays of rat embryo cells
(10). Such a list has been extended in recent years (Table 1).
Another line of work showed that oncogenes involved in the
transformation of avian hematopoietic cells also showed collab
orative effects (11).
The classification of oncogenes based on their collaborative
powers in transformation assays was paralleled in a striking
way by a totally distinct system of categorizing these genes:
classifying them by the intracci hilar localization of their respec
tive gene products. Those oncogenes that function like ras (by
collaborating with myc) encode cytoplasmic proteins while
those that function like myc (collaborating with ras) specify
nuclear proteins. This suggests that each group of oncoproteins
converges on a common target or pathway, one in the cyto
plasm, the other in the nucleus.
Not all oncogenes are seen to fit neatly into this scheme (1219). Thus, yet other pathways or targets may exist that are not
addressed by this ras/myc paradigm. Nonetheless, because of
this body of work, a terminology has evolved in which these
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ONCOGENES, ANTIONCOGENES, AND CARC1NOGENESIS
ke oncogenes encoding nuclear proteins are called ''nu
clear oncogenes" while these ros-like oncogenes specifying cytoplasmic proteins are called "cytoplasmic oncogenes."
The discovery of oncogene collaboration lent more substance
to the simple model of how multistep tumorigenesis works.
Restated, it would propose that each step in the tumorigenic
process reflects a mutation leading to the activation of one or
another cellular oncogene; the resulting activated oncogenes
then work together to induce the full neoplastic phenotypes of
the cell. Yet it must be said that fundamental aspects of this
model remain unproved. Is it true that multiple oncogenes are
invariably required to transform cells? And equally important,
how instructive is this model in understanding multistep carcinogenesis as it occurs in humans and in well-defined animal
models of tumorigenesis?
Apparent Violations of Multistep Carcinogenesis: Full Transfor
mation by Single Oncogenes
A number of experimental strategies would appear to violate
the rule that at least two oncogenes are required to transform
normal cells into fully malignant ones. To understand these, it
is necessary to go back and review how the ras/myc (or polyomavirus MT/LT or ros/ElA) oncogene collaborations were
first observed. Monolayer cultures of embryo cells (fibroblasts
or kidney cells) were transfected with individual oncogenebearing plasmici DNAs or cotransfected with several plasmids
carrying distinct oncogenes. When single oncogenes (e.g., ras
alone or myc alone) were transfected, the resulting monolayers
yielded few if any foci upon reaching confluence. However,
when two complementary oncogenes were cotransfected, foci
appeared, the cells of which proved tumorigenic upon inocula
tion into appropriate hosts (6-8). These were the results under
lying the conclusion that multiple oncogenes are required for
full transformation to tumorigenicity.
The simplest and most frequently observed deviation from
this scheme derived from transfection of ros-like oncogenes into
established cell types like Rat-1 cells or NIH3T3 cells (2, 20).
In this instance, single oncogenes indeed suffice to induce full
conversion to tumorigenicity in one step. Here the discrepancy
with the multistep model is easily rationalized. Such 3T3 cells
deviate from primary embryo cells in their established, immor
talized phenotype. This immortalization can be rationalized as
Table 1 Functional classification of cooperating oncogene in rat embryo
fibroblasts
Classification of oncogenes on the basis of their ability to collaborate in
transformation. For example, a ras oncogene can collaborate with mir, pS3,
protein or adenovirus EIA to transform rat embryo fibroblasts.
Cytoplasmic
oncogenes
Ha-roj
Ki-ras
N-ras
src (90)
Polyoma MT (6)
Nuclear oncogenes
myc (7, 8)
L-myc (93)
p53 (94, 95)
AdenoElA(8)
Polyoma LT (6, 7)
SV40LT (7)
Papillomavirus E7 (96)
In avian bone marrow cells
erbB
src
fps
mil
Hn-ras
ros
yes
sea
myc (11, 97)
myb
a premalignant phenotype similar to that induced by a myc
oncogene and known to make a primary cell responsive to
transformation by a ras oncogene acting alone. Viewed in this
way, established cells have already undergone changes reminis
cent of those induced by certain nuclear oncogenes. These
changes make these cells unsuitable for studying the full and
natural process of tumorigenesis that begins with a fully normal
cell.
Results like this refocus attention on primary cells that have
ostensibly undergone few if any changes in their growth control
mechanisms following their explantation from an embryo. They
should represent good models of the cells that suffer transfor
mation within a living tissue. Manipulations of such fully
normal cells make it clear that there are indeed conditions
under which single oncogenes, acting on their own, can induce
what appears to be total transformation. For example, when a
ras oncogene is cointroduced with a neomycin resistance marker
into embryo cells growing in monolayer, subsequent application
of neomycin results in killing off of the great majority of cells
in the culture and in the outgrowth of a small minority of
oncogene-bearing, neomycin-resistant transfectants. Their de
scendants growing to confluence form a monolayer of refractile
cells that are tumorigenic (9, 22).
A similar result is seen when embryo fibroblast monolayers
are infected with a retrovirus such as Harvey sarcoma virus,
which transduces a ras oncogene. If the virus is allowed to
spread through the monolayer, thereby infecting the great ma
jority of cells, then once again monolayers of fully transformed,
refractile cells soon appear which are also tumorigenic. In both
these instances, the cooperation of a second oncogene seems to
be gratuitous (23).
These results conflict with the model that requires at least
two oncogenes for transformation. The tension between the
two-gene model and these experimental results can be addressed
by introducing another element into the logic: the environment
of the oncogene-bearing cell. When monolayers of embryo cells
are transfected with a single oncogene (e.g., ras), only a small
number of cells initially acquire an oncogene, and each of these
cells is surrounded by normal untransfected neighbors. Under
these conditions, the occasional ros-transformants remain inapparent; they are unable to expand clonally to form visible
foci. In contrast, use of neomycin selection, as described above,
results in killing the normal neighbors, leaving only pure pop
ulations of ras transformants, the proliferation of which is now
unfettered. An analogous situation pertains upon high multi
plicity infection of monolayer cells by an oncogene-transducing
virus; the normal neighbors of an oncogene-bearing cell are no
longer present, having been recruited into the cohort of trans
formants through viral spread. The resulting pure populations
of transformants also grow aggressively.
All this suggests that neighboring normal cells exert a nor
malizing or inhibitory influence on the growth of ras transform
ants. When their presence and influence are absent, then pure
populations of ras transformants can proliferate to produce
large progeny clones. This makes the simple and important
point that the growth properties of a cell depend not only on
its own genotype (e.g., its complement of oncogenes) but on its
environment as well, a conclusion drawn by others from a
number of analogous experiments (24-30).
This point is made quite dramatically by recent /// vivo
experiments involving use of ros-transformed mouse skin keratinocytes. When a culture of these is mass transformed by high
multiplicity infection with Harvey sarcoma virus, then these
cells form rapidly growing squamous carcinomas upon implan-
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ONCOGENES, ANTIONCOGENES, AND CARCINOGENESIS
tation onto the back of a mouse. In contrast, when these same
cells are réimplantée)
together with a 4-fold excess of normal
dermal fibroblasts, then only small nonprogressing nodules are
observed (31). These dermal fibroblasts represent a cell type
with which keratinocytes normally coexist within the skin. Once
again the environment of oncogene-bearing cells constitutes a
strong determinant of their future growth properties. In these
several examples, normal, closely apposed cells strongly deter
the proliferation of ras transformants. An analogous result has
also been observed with wye-transformed cells (32, 33).
In Vivo Tumorigenesis with Single Viral Oncogenes
The above data suggest that a single oncogene, acting on its
own, is able to induce tumorigenesis under special conditions
in which the oncogene-bearing cell is isolated from the influ
ences of normal neighbors. Strong testimonial to this is also
provided by two decades of work on in vivo c;ire biogenesis using
a variety of rapidly oncogenic retroviruses, each of which trans
duces a single cellular oncogene in its genome. Included in this
list are the aforementioned Harvey sarcoma virus (ras), Abelson
leukemia virus («/>/),feline sarcoma virus (fes), and avian
myelocytomatosis viruses (myc) (34). In each case, the presence
of a coinfecting, replication-competent helper virus ensures that
these rapidly tumorigenic viruses are not only able to infect an
initially encountered target cell but also to spread centrifugali y
to all neighboring cells, recruiting them into the population of
transformants. This mimics the in vitro monolayer model in
which mass infection eliminates normal neighbors by recruiting
them into the cohort of transformants.
Here too, single oncogenes acting on their own appear suffi
cient for tumorigenesis. But appearances are misleading. The
large population of virus-infected transformants may well pro
liferate only for a limited time before a few of its progeny
sustain secondary changes that enable them to grow as truly
autonomous, malignant cells. This possibility can be addressed
by analysis of the clonality of tumor cell populations emerging
from such virus-inoculated tissues. Discovery of polyclonal
populations would indicate that many if not most of the initially
transformed cells can be tumorigenic without requiring rare
secondary changes; mono- or oligoclonality would indicate that
only a few of the initially large number of transformants acquire
the secondary changes necessary to propel them to a tumori
genic state. A recent study indicates that in one case such
tumors are indeed mono- or oligoclonal and that the initial
burst of proliferation seen after mass infection by a transform
ing retrovirus may not suffice to create a stably growing tumor
mass (35). The nature of such secondary changes is presently
obscure; their implied existence suggests that even upon mass
transformation of a cell population by a single oncogene, sub
sequent events must intervene before the cells are genuinely
malignant.
Mouse Models of Tumorigenesis
Oncogene-mediated transformation studied in vitro acquires
validity only if it reflects aspects of tumorigenesis as it occurs
in vivo. In vitro transformation can indeed mimic certain early
steps in tumor formation by creating cells of a similar genotype.
For example, strong evidence has been reported which shows
that the initiating event in mouse skin carcinogenesis is the
creation, through chemically induced mutation, of an activated
ras oncogene. This genotype can be mimicked readily through
use of transducing retroviruses which serve to convey an exog
enous ras oncogene into an apparently small proportion of
keratinocytes infected in situ in the skin. The small minority of
infected cells function as if they has been altered by an initiating
carcinogen, yielding papillomas upon treatment by a tumor
promoter (36).
The proportion of cells infected by such a transforming
retrovirus in a target tissue would seem to be a critical parameter
in virus-induced tumor initiation. Nonviral tumors start out as
small nests of partially transformed cells surrounded by a large
amount of normal tissue, a state which would appear to be
mimicked by infecting scattered skin keratinocytes with Harvey
sarcoma virus. A contrasting situation results upon efficient
mass infection and transformation of all the cells in a tissue.
This must drastically alter an important element in the dynam
ics of tumorigenesis by depriving initiated, oncogene-bearing
cells of close contact with normal neighbors. Such a large mass
of initiated cells may quickly undertake clonal expansion since
they are no longer confronted with the inhibitory influences of
normal tissue.
This notion has important bearing on another frequently
used model of tumorigenesis, that involving transgenic mice.
As many as 20 different mouse lines have been derived over the
past 5 years in which activated oncogenes are inserted into the
mouse germ line (37). Invariably, the expression of these on
cogenes is driven by a tissue-specific transcriptional promoter
that may also be regulated in a stage-specific manner. The result
is usually the appearance of a tumor at a site that is predicted
by the nature of the promoter chosen to regulate oncogene
expression. Thus, the mouse mammary tumor virus transcrip
tional promoter engenders largely mammary tumors while the
insulin gene promoter favors pancreatic tumors.
By creating cohorts of mice with well-timed onsets of pre
dictable tumors, these transgenic models would seem to provide
ideal experimental models of spontaneous tumorigenesis. But
they fall short in one important aspect. Instead of creating
small, isolated nests of initiated, oncogene-bearing cells, these
transgenes create tissues in which virtually all the cells are
expressing an activated oncogene. In so doing, the transgenic
model fails to address one of the most important aspects of
tumorigenesis, i.e., the interactions of transformants with their
normal neighbors during the early stages of this process.
Early Steps: Transcending a Hostile Environment
Repeated mention has been made here of a critical early step
in tumorigenesis, the process by which a small, early preneoplastic cell clone expands in spite of the inhibitory influences
of normal neighbors. Contrary to earlier discussion in which
ras and myc oncogenes were given equal weight, the attention
in this discussion has been focused on the role of ras oncogenes
in affecting the behavior of early tumor cell clones. The bias is
intentional in that activated ras oncogenes have been found in
a number of preneoplastic murine and human tumor models
including those of the skin, colon, and hematopoietic system
(38-40). This suggests that ras activation is often a relatively
early event in tumor formation.
How are isolated premalignant ras transformants normally
able to expand to the large clonal sizes that then permit the low
probability secondary changes necessary for truly autonomous
neoplastic growth? Clearly in the vast majority of cases, ras
transformants fail to do so, remaining as isolated single cells
or small pockets of cells that are hemmed in by their environ
ment. This point is made most dramatically by calculating how
many cells in the human body have through accidents of DNA
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ONCOGENES, ANTIONCOGENES, AND CARCINOGENESIS
replication acquired activating oncogenic point mutations in
their ras genes. The number, variously calculated at 105-106,
vastly exceeds the number of observable premalignant lesions
any one of us will experience in a lifetime.
An escape from environmental inhibition can be forced ex
perimentally through use of tumor promoters. To cite the
previously mentioned case, small nests of initiated rax trans
formants can be driven into macroscopic papillomas by TPA.3
By the present logic, this tumor promoter contributes to skin
tumorigenesis through its ability to allow ros-bearing keratinocytes to transcend the growth-inhibitory influences of their
normal neighbors, including perhaps those of the dermal libro
blasts to which they are closely apposed within the skin. Anal
ogously, in the rat embryo fibroblast monolayer model, TPA
application enables the focal outgrowth of ras transformants,
the presence of which would otherwise be inapparent (41). This
promoter acts as an agent that confers special growth advantage
on ros-bearing cells; conversely, the initiated, ros-bearing cells
behave as if they were especially responsive to the growthstimulatory influences of the promoter. A similar suppression
of growth by normal neighbors and TPA-induced reversal of
this suppression has been seen with UV-irradiated C3H lOT'/z
mouse fibroblasts (30).
It is unclear how TPA acts to propel forward the growth of
the raj-bearing cells described above. One obvious answer
comes from the observations that gap junction communication
between normal and tumor cells correlates with the susceptibil
ities of tumor cells to inhibition by their normal neighbors, and
the fact that TPA dramatically reduces the gap junction-me
diated communication that normally couples the metabolism
of neighboring cells (42-45). In so doing, the TPA may interdict
the flow of growth-inhibitory signals from normal cells to an
initiated neighbor, thus giving the latter a free hand to prolif
erate (32, 33, 42-46). While this mechanism may explain some
of the effects of TPA, I suspect that the biological reality is
more complex. The kinase C enzyme activated by TPA sits
astride a central mitogenic pathway within the cell. Thus, TPA
would seem to provide a strong growth impetus to a ras-bearing
cell in addition to liberating it from the inhibitory influences of
its environment.
Later Steps: Collaborating Secondary Genetic Changes
The scenario of tumor evolution drawn here echoes schemes
widely accepted in the field of carcinogenesis. Initiating carcin
ogens create a critical genetic change (in this case involving a
ras gene); any resulting initiated cells expand clonali y under the
influences of a tumor promoter (e.g., TPA) until they form a
large enough clone of descendants to permit the occurrence of
low probability, secondary genetic changes; and these secondary
changes create alÃ-elesthat collaborate with the initially induced
ras oncogene to produce a fully tumorigenic cell that is no
longer dependent on the promoter for its continued growth
(47-49).
Missing from such a scheme is a clear understanding of the
nature of the genes that are activated late in tumorigenesis
during progression and serve as collaborators with the oncogenes created initially. Obvious candidates for these collabora
tors are the myc gene and analogously acting "nuclear onco
genes" like N-myc, L-myc, and p53 (Table 1; Ref. 50). These
all act synergistically in the in vitro transformation assay with
ras-like oncogenes. One or the other of these, activated during
3The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate;
pl05-Rb, M, I05.000 Rb gene-encoded protein.
tumor progression, could well serve as the second gene required
for autonomous neoplastic growth.
Regrettably, faith in such a model is not reinforced by much
data collected over the past 5 years. Very few tumors have been
found that carry both activated ras oncogenes and aberrations
of nuclear oncogenes like myc. A first reaction to such failure
is that the scheme of ras-myc collaboration is incorrect and
does not address the biological realities of tumor cell biology.
However, I suggest that a more useful recourse is to restate the
lesson of ras/myc collaboration in a more general form: that
the conversion of a normal cell into one that is fully tumorigenic
involves at least two types of change in cell physiology, one
occurring in the cytoplasm and one in the nucleus. The cytoplasmic changes may be induced by ras or other analogously
acting oncogenes; those in the nucleus may be achieved by myc
or other genetic changes that create phenocopies of the mycinduced state. These required nuclear changes may depend on
genes that operate on totally different principles than those of
the well-characterized nuclear oncogenes.
A similar dilemma is posed by the existence of spontaneously
immortalized cells (e.g., NIH3T3, Balb/c3T3, C3H lOT'/z)
which ras can readily transform to a tumorigenic state. These
cells appear to lack wye-like oncogenes that might mediate
their immortalization and ras responsiveness. Perhaps their
myc-like phenotypes may also be achieved through mechanisms
that do not depend upon the mutation of nuclear oncogenes
like myc.
The Search for myc Surrogates
What clues do we have in the search for the elusive genetic
changes that can mimic physiologically the activation of nuclear
oncogenes? This question can be approached by examining the
physiological effects of myc on the cell, myc is an immortalizing
gene, but unexpectedly this trait may not be central to its role
in tumorigenesis. The relationship of immortalization in tissue
culture to in vivo growth properties of tumor cells is at best
obscure. Many biopsied tumor cells are not immortal in culture.
Moreover, it has been shown that immortalization of cells in
culture does not necessarily confer responsiveness to the trans
forming effects of oncogenes like ras (51). By this logic, the
abilities of myc (or El A) to confer immortalization and respon
siveness to ras are physiologically separable qualities and it is
the latter quality (ras responsiveness and associated oncogene
collaboration) that is more central to tumorigenesis.
What then is the essence of the collaborating powers of myc
(or myc-like oncogenes)? Here, once again, the embryo fibro
blast monolayer assay proves instructive. If ray-transformed
cells are unable to induce foci in the presence of normal neigh
bors while ras+myc cells growth strongly, then one mechanism
of action of myc is clear: myc enables ras transformants to
ignore or override the inhibitory influences of normal neigh
boring cells.
How myc oncogenes can do this mechanistically is not at all
apparent. One clue may derive from the peculiar behavior of
myc and the group of other nuclear oncogenes that it represents.
Expression of the normal (protooncogene) versions of these
genes is highly regulated and almost always positively correlated
with growth, myc itself is expressed at low basal levels in
quiescent fibroblasts and its expression is substantially in
creased and maintained upon entrance into the active cell cycle.
myc and other nuclear oncogenes are turned on rapidly in
response to a number of cell mitogens.
This provides a clue and a speculation; perhaps equally
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ONCOGENES, ANTIONCOGENES, AND CARCINOGENESIS
important negative regulatory mechanisms also govern nuclear
protooncogene expression. Perhaps antimitogenic signals im
pinging on the cell actively suppress the expression of myc-like
genes. Such negative environmental influences may encompass
signals involved in maintaining contact inhibition, signals
through which growth-inhibitory hormones like interferon and
transforming growth factor ßshut down cell growth, indeed
even the signals used by normal cells to shut down the growth
of ras transformants. While poorly documented, these inhibi
tory mechanisms may all operate through their ability to sup
press expression of /wye-like genes including myc itself (52-56).
The genetic changes that create the constitutively expressed
myc oncogene may serve to uncouple the regulation of this gene
from the environmental influences that usually act to shut down
the normal myc gene. From the viewpoint of cell physiology,
such uncoupling of nuclear genes from extracellular signals,
achieved through their genetic alteration, may create the same
end results as are seen upon uncoupling of cell-to-cell contact
by TPA.
One often depicts myc oncogenes as constitutively expressed
and in this way no longer dependent on the mitogenic stimu
lation normally required to elicit and maintain their expression
(57). Perhaps we should think of myc oncogenes in an entirely
different light: as genes that can remain on in spite of the
presence of antimitogenic influences that normally operate to
shut down expression. This translates further into possible
insight as to how myc oncogenes enable ras transformants to
ignore their normal neighbors; the myc oncogene, unlike its
normal protooncogene, can remain on in spite of extracellular
influences that would normally force its shutdown.
Creating Constitutively Activated myc-like Genes
We know rather little about how nuclear protooncogenes are
regulated either positively or negatively. A number of research
groups have focused on the DNA sequences that are linked to
their transcriptional promoters and serve to increase use of
these promoters. Virtually nothing is known of the m-acting
sequences that allow the cell to repress expression of these
protooncogenes. Clearly by deleting or inactivating negatively
acting regulatory sequences of these genes, one may achieve
many of the conditions required for their constitutive expres
sion, thereby making these genes into oncogenes (58, 59).
An alternative molecular mechanism may achieve the same
end result. Rather than deleting the as-acting regulators of
expression, other types of damage may serve to delete from the
cell the diffusible trans-acting factors that normally act to
mediate shutdown of genes like myc. This idea deserves gener
alization: a phenocopy of the myc oncogene-induced state may
be achieved by knocking out the signal transducing pathways
that normally shut down cell growth in response to environ
mental growth-inhibitory influences. The lost elements of these
signaling pathways may be either regulators of nuclear protoon
cogene expression or even the targets of nuclear oncoprotein
action.
Such an idea forces the restructuring of the concept of on
cogene collaboration. The events that often serve to collaborate
with ras activation may not be the activations of nuclear onco
genes; instead, the inactivation of negative regulatory pathways
and the genes that encode them may be the most frequent way
of achieving the same end result. After all, it is usually far easier
to knock out gene function than to create the hyperactive alÃ-eles
that we recognize as oncogenes.
Tumor cell genomes may often contain activated oncogenes
coexisting with inactivated versions of negative regulatory
genes, the two sets of changes collaborating to confer iumorigenicity. In the case of many raj-bearing tumors observed to
date, complementary wye-like changes may be sustained
through gene inactivation. Moreover, immortalization/estab
lishment in vitro and the acquisition of ras responsiveness may
also be associated with the loss of negative regulatory genes
(60).
Tumor Suppressor Genes: Dominance and Recessiveness
The existence of such negative regulatory genes is suggested
by an extensive literature describing the consequences of fusing
normal cells with tumorigenic cells (61-65). A frequent out
come is the loss of tumorigenicity by the hybrid cell. This
supports the notion that the creation of the tumorigenic state
often involves loss of growth-regulatory genes that are restored
to the tumor cell upon fusion with a normal partner. These lost
negative regulators may be termed tumor suppressor genes in
the sense that they serve to regulate normal cell growth; their
loss removes a critical constraint on proliferation that in turn
can contribute to tumorigenicity.
Some have viewed the nontumorigenic phenotype of these
hybrids as evidence that the genes responsible for the tumor
phenotype act recessively, being unable to elicit tumorigenicity
in the presence of the wild-type intact alÃ-elesthat act dominantly. But this interpretation is unwarranted. If tumorigenesis
is dependent on mutations in a number of critical genes, some
of the mutations may create dominant, deregulated alÃ-eles,
while other essential mutations in the same cells may create
recessive, inactivated alÃ-eles.If only one of the essential, con
tributory steps in tumorigenesis involves the creation of inac
tive, recessive alÃ-eles,then the phenotype of tumorigenicity as
a whole will behave as if it too were recessive. As a consequence,
it has little meaning to speak of tumorigenicity as a dominant
or recessive phenotype. One can only use these terms meaning
fully to describe the behavior of specific gene alÃ-elesacting in
the presence of their wild-type homologues.
To cite an example, in certain cases a ras oncogene-containing
cell has been reverted to nontumorigenicity following fusion
with a normal partner (65, 66). Here the interpretation has
been that the ras oncogene acts recessively, i.e., that the normal
cell contains gene products that override and suppress the
activity of ras (63, 66). But if the malignant state of the rasbearing cell depends both on the activation of a ras oncogene
and on the inactivation of a negative regulatory gene, then such
interpretation are untenable. The dominance or recessiveness
of a ras oncogene can be known only by gauging its activities
relative to those of its wild-type alÃ-ele.And the reversion of
these cells to a nonmalignant state may only signal the essential
involvement of other genes, fully unrelated to ras, the inacti
vation of which acts collaboratively with ras activation to create
the end point of tumorigenicity. The isolation of one such
candidate gene, which partially suppresses the malignant phe
notype of a ras-transformed cell, has recently been reported
(67).
Molecular Nature of Tumor Suppressor Genes
The use of somatic cell hybridization has served well to point
out the existence of tumor suppressor genes but has proved to
be a cumbersome tool in learning more about them. In order to
understand these genetic factors, we need to identify these genes
with discrete genetic loci, to isolate these as molecular clones,
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ONCOGENES.
ANTIONCOGENES,
and to study the biochemical modes of action of their encoded
proteins.
The Rb gene, the loss of which predisposes to retinoblastomas
and osteosarcomas, has proved to be the most tractable of a
possibly large class of such genes. A conceptual breakthrough
in our understanding of retinoblastoma origins came from the
hypothesis of Knudson (68, 69) that all retinoblastoma tumors
must sustain mutations in two distinct genes prior to tumor
development. In 1979, Yunis and Ramsay (70) provided the
first evidence that at least one of these mutations creates inactive
alÃ-eles.This was suggested by their observation of the occasional
deletion of genetic material on chromosome 13ql4 in retino
blastomas. This suggested further that this chromosomal region
harbored a discrete gene (i.e., Rb) that served repeatedly as the
target of genetic inactivation events occurring during the for
mation of these tumors.
The identity of the second of Knudson's hypothesized target
genes was soon apparent; it was the surviving, hitherto intact
copy of the Rb gene on the homologous wild-type chromosome.
Study of a closely linked chromosomal marker gene, esterase
D, showed that its heterozygosity, often observed in the normal
tissues of a retinoblastoma patient, was reduced to homozygosity in the tumor cells (71, 72). By analogy, the implications for
Rb were clear; the second step in tumorigenesis was the loss of
the surviving intact alÃ-eleof the Rb gene, it being replaced by a
copy of the initially mutated one. This established the point
that both copies of the Rb gene need to be lost or inactivated
in order for phenotype to be affected. In this sense the Rb gene
shows the properties of a negative regulatory gene of the type
described earlier.
A similar phenomenology has been derived for the Wilm's
locus, in which interstitial deletions and reductions to homo
zygosity have been observed associated with a chromosome 11
locus (73). Moreover, the paradigm has been generalized
through use of polymorphic restriction enzyme site markers in
which a number of distinct tumor types have been associated
with reductions to homozygosity of particular chromosomal
loci (74). In most of these cases, direct evidence of gene inacti
vation is still lacking and the recessiveness of the involved
alÃ-elesis imputed from the observed homozygosities.
In the case of the Rb locus, a candidate gene has been isolated:
it is a 190-kilobase stretch of chromosome 13ql4 that is ex
pressed in many tissues and specifies the structure of a M,
105,000 phosphoprotein (75-78). The importance of this pro
tein, or rather its loss, to the genesis of retinoblastoma is
underscored by recent evidence which shows that this protein
is present in normal retinoblasts but absent from 16 of 16
retinoblastomas.4 To date the Rb gene stands as the only
AND CARCINOGENESIS
bution of retinoblastoma incidence. This type of tumor has been
observed to occur spontaneously only in our own species, yet
the gene is present and apparently active in all mammals.
The Function of the Rb Protein
The central issue surrounding the Rb gene is the function of
its encoded protein, pl05-Rb. How do its properties fit with
the model of gene inactivation and wye-like phenotypes drawn
above? While little is known about the biochemistry of the Rbencoded protein, its involvement in the physiology of growth
regulation has been highlighted by a dramatic discovery made
in the spring of 1988. This discovery directly ties the Rb protein
to the multistep pathways of transformation discussed here.
Specifically, it places the Rb protein in the middle of the arena
of nuclear, wye-like oncogenes.
The work originated in the laboratory of Ed Harlow of Cold
Spring Harbor who together with others had shown that the
oncoprotein encoded by the EIA oncogene of human adenovirus type 5 is found complexed with a variety of host cell proteins
in virus-transformed cells (85, 86). These proteins number more
than six. Their multiplicity may provide a molecular explana
tion for the multiple functions exerted by the EIA oncogene,
which include cell immortalization, oncogene collaboration,
and regulation of transcription of a number of viral and host
genes. Each of the host cell target proteins to which EIA
complexes may represent a regulator of a distinct cellular
pathway, the activities of which may be modulated following
complex formation.
One of these host cell target proteins was found to be p 105Rb (87). This association is not merely adventitious, since
mutations that inactivate the transforming activities of EIA
also knock out its ability to complex with pl05-Rb. Since
inactivation of the Rb gene and resulting loss of pl05-Rb is
critical to the formation of retinoblastomas, it is tempting to
speculate that a similar result is achieved epigenetically through
the ability of the EIA oncoproteins to complex with and func
tionally inactivate the AA-encoded protein.
This strengthens the hand of those who would call Rb an
"antioncogene." Here we see a direct physical confrontation
between an oncogene product and that of the Rb gene. Never
theless, the term is misleading in that it implies that the role of
Rb is to antagonize oncogene function. More palatable is the
term "tumor suppressor." Ultimately the term "growth sup
pressor" may be seen to mirror most closely the normal func
tions of the Rb and its analogues.
Other complexes similar to Rb:ElA have been discovered
more recently. They are found between Rb and oncoproteins of
example of a potentially large class of genes that has yielded to at least two other DN A tumor viruses, those of the SV40 large
T (88) and human papilloma type 16 E7 (89) oncogene.
molecular cloning.
Through an apparent process of convergent evolution, three
The isolation of the Rb gene leaves a number of unresolved
different groups of DNA tumor viruses (adeno-, papilloma,
questions and paradoxes. The most direct proof that the cloned
gene is indeed the Rb gene must come from introduction of a SV40) have developed oncoproteins that specifically complex
with pl05-Rb. In the case of SV40 large T, a point mutation
cloned intact copy into retinoblastoma cells with observed
restitution of normal growth control. A recent report of this affecting only 1 of the 708 amino acid residues of the protein
has appeared in the literature (79). The cloning of this gene inactivates its transforming powers and at the same time de
stroys its ability to form complexes with pl05-Rb (88). This
also reveals apparent paradoxes. While the gene is expressed in
argues that these associations are central to the ability of these
a rather wide array of tissues, ostensibly participating in their
oncogenes to contribute to transformation.
growth control, its inactivation seems to lead to only a narrow
All this highlights the unexpectedly central position that
range of tumor types, notably retinoblastomas, soft tissue and pl05-Rb
occupies in the growth-regulatory circuitry of the cell,
osteosarcomas, small cell carcinomas of the lung, and bladder
being involved in a number of distinct mechanisms of transfor
carcinomas (80-84). Equally perplexing is the species distrimation. Of equal interest is the biochemical basis that this
4 J. Horowitz and R. A. Weinberg, manuscript in preparation.
provides to mechanisms of oncogene collaboration. All three of
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ONCOGENES,
ANTIONCOGENES,
the oncogenes have been shown to function as myc-\ike genes,
each able to immortalize and to collaborate with ras oncogenes
in transformation of primary cells (Table 1). The oncogene
collaboration test, described earlier, has been a purely functional
means of classifying oncogenes. It is satisfying that these on
cogenes, functionally allied through this test, can now be par
tially understood in terms of a shared molecular mechanism.
The interactions of these three different DNA virus oncoproteins with a common cellular target suggest that they act by
mimicking an endogenous cellular protein. An attractive can
didate would be the cellular myc protein itself, but the evidence
is still lacking.
These interactions provide support for the notion described
earlier that inactivation of a tumor suppressor gene yields
phenotypes that resemble those resulting from myc-tike onco
genes. How physiological changes like immortalization and
oncogene collaboration are achieved at the molecular level
remains unclear. It is tempting to speculate that the Rh gene
product is a component of a growth-inhibitory signalling chain.
When the Rb protein is lost through alteration of its gene, the
responsiveness of the cell to negative signals may be compro
mised or lost. Similarly, by complexing with the Rb protein,
DNA virus oncoproteins may inactivate its function and in this
way deprive the cell of a vital link needed to transduce growthinhibitory signals.
Oncogenes and Antioncogenes in Multistep Carcinogenesis
These models and recent findings have implications for the
genesis of retinoblastoma tumors. It has been argued that
inactivation of both copies of the Rb gene suffices to create
these retinal tumors (68, 69). But ifRb inactivation creates only
/wye-like changes in the cell, then it may be the case that
complementary changes in a cytoplasmic oncogene may be
required to create a truly autonomously growing tumor. Such a
notion is not yet addressed by direct experimental studies of
retinoblastoma genomes.
Another type of tumor cell genome, that of colon carcinoma
cells, has already yielded evidence of activated ras oncogenes
occurring together with reductions to homozygosity of loci on
chromosomes 17 and 18 (40). The inactive state of these homozygous alÃ-eleshas not yet been shown. Nonetheless, this appears
to provide an attractive tumor model in which oncogene acti
vation and antioncogene inactivation collaborate to create the
full malignant phenotype.
Where will all this take us? It should be clear that oncogenes
present only part of the answer to the puzzle of multistep
tumorigenesis. The vista of the negative genetic regulators of
cell growth, termed variously antioncogenes, tumor suppressor
genes, or growth suppressor genes, is just opening. These genes
will be hard to find and study since their existence is most
apparent when they have undergone inactivation. However, the
struggle to find them will be worth the effort. They will tell us
as much about cancer as oncogenes, perhaps even more. And
they will fill a large gap in our current picture of the growthregulatory circuitry of the cell.
Acknowledgments
The author would like to thank Ehry Anderson for excellent help in
preparing this manuscript. He is an American Cancer Society Research
Professor.
AND CARCINOGENESIS
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Oncogenes, Antioncogenes, and the Molecular Bases of
Multistep Carcinogenesis
Robert A. Weinberg
Cancer Res 1989;49:3713-3721.
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