Transformation and Oncogenesis

Lecture 18
Biology W3310/4310
Virology
Spring 2016
Cause and effect, means and ends, seed
and fruit, cannot be severed; for the effect
already blooms in the cause, the end pre-
exists in the means, the fruit in the seed.
RALPH WALDO EMERSON
Transformation

Principles of Virology, ASM Press

The puzzling properties of transformed
cells in the laboratory

• Immortal: Grow indefinitely (HeLa)

• Loss of anchorage dependence
• Loss of contact inhibition
• Colony formation in semi-solid media
• Decreased requirements for growth factors (serum)
 Oncogenesis
• Development of cancer

- Tumor: swelling caused by abnormal growth of tissue,

benign or malignant

• Cancer is a genetic disease

• 8.2 million deaths/yr developed countries

• Mutations (~12) affect signal transduction pathways that

govern cell proliferation, survival, determination of cell fate,
maintenance of genome integrity

• Mutations may be inherited, caused by DNA damage,

environmental carcinogens, infectious agents including
viruses
 Transformation and oncogenesis are
distinct

May not be Requires additional genetic changes

oncogenic

• Studying virus-transformed cells provides insight

into molecular events that establish oncogenic
potential

• No virus can do it all

 Human cancer viruses
Virus Cancer
Burkitt’s lymphoma
Epstein-Barr virus
Nasopharygeal carcinoma
Hepatitis B virus Hodgkin’s lymphoma
Hepatocellular carcinoma
Hepatitis C virus Hepatocellular carcinoma
Human T-lymphotropic virus-1 Adult T cell leukemia
Human immunodeficiency virus-1
Cervical, penile, anogenital, head
Human papillomavirus
and neck cancers

Kaposi’s sarcoma
Kaposi’s sarcoma herpesvirus Primary effusion lymphoma
Multicentric Castleman’s disease

Merkel cell polyomavirus Merkel cell carcinoma

Contributing factor in ~20% of human cancers

 Virus-induced cancer

Transformation and oncogenesis is not required

for replication of any* virus
On October 1, 1909, Dr. Peyton Rous removed a tumor
from an English hen and injected a cell-free filtrate from
the tumor into another healthy chicken, which later
developed the same type of tumor

Cancer could be caused by a viral infection!

 It took the world almost 50 years to accept this idea

Dr. Rous lived long enough to be awarded the

Nobel Prize for Physiology and Medicine in 1966
for his research

His legacy: RSV; Rous Sarcoma Virus,

a key player in two more Nobel Prizes
 “By the 1950s, cancer researchers had
split into three feuding camps.

The virologists, lead by Rous, claimed

that viruses caused cancer, although no
such virus had been found in human
studies.

Epidemiologists...argued that
exogenous chemicals caused cancer,
although they could not offer a
mechanistic explanation.

The third camp possessed weak,

circumstantial evidence that genes
internal to the cell might cause cancer...
Scribner, 2011

http://www.pbs.org/kenburns/cancer-emperor-of-all-maladies/home/
 Howard Temin

In 1951, a young virologist named Howard Temin

arrived at Cal Tech to study the genetics of fruit flies.
Restless and imaginative, he soon grew bored with
fruit flies. Switching fields, he chose to study Rous
sarcoma virus in Renato Dulbecco’s laboratory.

Until the late fifties, Rous sarcoma virus had been

shown to cause tumors only in live chickens. Temin
imagined creating cancer in a petri dish. In 1958, in
his seventh year in Dulbecco’s lab, Temin succeeded.
Scribner, 2011
He added Rous sarcoma virus to a layer of normal cells in a petri dish. The infection
of the cells incited them to grow uncontrollably, forcing them to form tiny distorted
heaps containing hundreds of cells that Temin called foci. The foci, Temin reasoned,
represented cancer distilled into its essential, elemental form: cells growing
uncontrollably, unstoppably - pathological mitosis.

Temin went on to discover RT in RSV

Avian cells transformed by RSV
Fusiform Round, refractile

Scribner, 2011 Principles of Virology, ASM Press

 Transforma)on of cells by viruses

• 1962: A4er infec;on with polyomavirus, rare BHK21

cells changed shape, kept growing
• 1964: A4er infec;on of Swiss 3T3 cells with SV40, rare
cells grew as colonies

Most of the infected cells died, but rare cells did not

They were “transformed”

 How can a viral infection transform a
cell?

• Cytopathic effects must be reduced or

eliminated
- The infected cell does not die
• Viral replication must be reduced or eliminated
- Transformed cells do not produce virions
• The cell must continue to divide
- It becomes immortal
 Go to:

m.socrative.com
room number: virus
Which of the following is not a property of
transformed cells?

1. Increased requirements for growth factors

2. Immortality
3. Loss of anchorage dependence
4. Loss of contact inhibition
5. Colony formation in semi-solid media

1
 Route to understanding viral
transformation of cells in culture and
relationship to cancer was convoluted

(1900s) Retroviruses (1943) in vitro (1920s) DNA Viruses

cancer biology

(1950s) in vitro Convergence (1959) in vitro

studies with RSV (1960s, 1970s) studies with
polyomavirus

(present) Unified theory of cell growth control

Principles of Virology, ASM Press
 How does Rous sarcoma virus cause
tumors in chickens and transform cells in
vitro?

(1900s) Retroviruses (1943) in vitro (1920s) DNA viruses

cancer biology

(1950’s) in vitro Convergence (1959) in vitro

studies with RSV (1960s, 1970s) studies with
polyomavirus

(present) Unified theory of cell growth control

Principles of Virology, ASM Press
 Avian leucosis retroviruses (ALV) are
endemic in virtually all chicken flocks

• Ellerman & Bang 1908

• Most chickens infected with ALV within a few months of

hatching

• Leucosis (leukemia) occurs sporadically in infected birds >14

wk old (3%)

• 97% of birds have transient viremia, become immune, don’t

get leukemia Principles of Virology, ASM Press
Infected birds develop other cancers as
they age

Rous was lucky!

• Connective tissue tumors or sarcomas (solid tumors)

• Virus isolated from these solid tumors rapidly cause
sarcomas, not leucosis

• Rous isolated one of these viruses: Rous sarcoma virus,

RSV

• Most of these sarcoma viruses are defective

 How does RSV, but not ALV, cause
sarcomas?
• Key finding: the viral genomes from solid tumors
were recombinants!

• A piece of the ALV genome is replaced with a

segment of host DNA called an oncogene

J. Michael Bishop and H. Varmus iden)fied the oncogene (v-SRC)

carried by Rous sarcoma virus in 1976

Nobel Prize to both in 1989 for this discovery

 Major insight
• ALV infected birds came down with a variety of
tumors

• These rare tumors all contained retroviruses

derived from ALV, but most were defective and all
were different

• Rous was lucky - his RSV isolate was not defective

The retrovirus genomes isolated from each new solid tumor
had different host DNA, NOT the v-SRC gene found in RSV

Each new DNA segment had a novel chicken oncogene

A gold mine for molecular oncology

Genomes of transducing retroviruses

Principles of Virology, ASM Press

Defective vs non-defective retroviruses

• Defective viruses require helper virus to produce

more virus

• Usually missing envelope proteins

• Envelope genes deleted during oncogene capture

Principles of Virology, ASM Press

Mechanism for oncogene capture

Principles of Virology, ASM Press

 Proto-oncogenes

• >60

• In all cells, control cell growth

• Highly regulated

• Normal cellular genes abbreviated as c-ONCS,

eg c-SRC, c-MYC, c-MOS, C-RAS

• Certain retroviruses isolated from tumors carry

altered copies of c-ONCS abbreviated as v-
ONCS, eg v-SRC, v-MYC, v-MOS, v-RAS
Subcellular location of major classes of
oncoproteins
Growth Factors
sis

erbB, fms, kit, ros, sca abl, src, yes

Growth factor receptors Membrane-bound ras
protein kinases G-proteins Original phenotype in
cultured cells:
Cytoplasmic
protein
erbA, ets, fos, jun, kinases TRANSFORMATION
myb, myc, rel, ski
fps, mos, raf
Nuclear oncogenes,
transcrip)onal regulators, Uncontrolled cell
cell cycle regulators growth
The cell cycle
Mitogenic signals
revealed by studying
transforming retroviruses
Dominant oncogenes

Principles of Virology, ASM Press

 Retroviruses transform cells by three
mechanisms
• Rapid tumor formation: eg RSV; 2 weeks
- RSV has activated dominant oncogene in genome (v-SRC)
- Protein produced immediately when virus replicates
• Intermediate kinetics of tumor formation: eg ALV;
months
- ALV carries no dominant v-ONC gene
- cis-activation: provirus turns on expression of endogenous oncogene

• Slow kinetics of tumor formation; eg HTLV; years

- HTLV carries no dominant v-ONC gene
- Does not cause cis-activation of local oncogenes
- A viral regulatory protein activates oncogenes by trans-activation
Proviruses with different transforming
potential
Rapid

Intermediate

Slow

e.g., IL2 and the IL2 receptor

 Mammalian transforming retroviruses

• Retroviruses transform cells as a mistake or

byproduct of their life cycle - they must
integrate their DNA

• No obvious viral requirement for

transformation or for oncogenesis

Walleye dermal sarcoma virus

Principles of Virology, ASM Press
 Go to:

m.socrative.com
room number: virus

Which of the following allows Rous sarcoma virus to

transform cells?

1. Presence of the env gene

2. Presence of a pol gene
3. Presence of a src gene
4. Presence of LTRs
5. None of the above
2
How study of DNA virus transformation
revealed how cell cycle is regulated

(1900s) Retroviruses (1943) in vitro (1920s) DNA Viruses

cancer biology

(1950s) in vitro Convergence (1959) in vitro

studies with RSV (1960s, 1970s) studies with
polyomavirus

(present) Unified theory of cell growth control

Principles of Virology, ASM Press

 DNA tumor viruses

First oncogenic DNA virus discovered was

papillomavirus that causes warts (papillomas) in
cottontail rabbits - Richard Shope, 1933
 DNA tumor viruses: Polyomaviridae

• Ludwig Gross discovered murine polyomaviruses

in 1953

• Caused rare tumors under certain conditions

- Natural host is the mouse
- Ubiquitious in mice; no role in mouse cancer
- Makes tumors of many tissues (poly-oma) in
infant hamsters, rats, rabbits
 DNA tumor viruses: Polyomaviridae

• Eddy and Hilleman showed that SV40, a

contaminant of early poliovirus vaccines, induced
rare tumors in newborn hamsters - 1962

• Several million Americans were infected with SV40

by poliovirus immunization

- Natural host is monkey

- Causes no tumors in monkeys
- Does not transform monkey cells in culture
Response of different cells to infection

Species SV40 Mouse polyomavirus

Monkey Permissive Non-permissive

Mouse Non-permissive Permissive

*Hamster Semi-permissive Semi-permissive

*Rat Semi-permissive Semi-permissive

* Tumors
Polyomaviral transformation of cultured
cells is rare

• 1 transformed cell per 100,000 infected cells

• Why is it so rare?

• How does this property relate to rare tumor

formation in animals?
 Adenoviridae: Another family of
transforming DNA viruses

• Many human serotypes, do NOT cause cancer

in humans

• Ad 12-18 tumors in hamsters

• Ad 7-11 poorly tumorigenic in hamsters

• Tumors and transformation of cells: like

polyomaviruses and papillomaviruses, very
rare events
Principles of Virology, ASM Press
Key finding: Viral T antigens in tumors
and transformed cells

• SV40: Large T, small T

• Polyomaviruses: Large T, middle T, small T
• Papillomaviruses; T encoded by E5, E6, E7 genes
• Adenoviruses: T antigens are E1A, E1B

All different proteins!

Principles of Virology, ASM Press
T antigens are encoded by essential viral
genes

• Required for replication

• Activate viral transcription

• Required for viral DNA synthesis

• Only viral genes always retained in tumor cells

or transformed cells

• T antigen alone can transform cultured cells

Principles of Virology, ASM Press

 Three seemingly unconnected discoveries in DNA
virus biology were critical to understanding the link
between viruses, transformation, and the cell cycle

1. 53 kDa cell protein binds SV40 T antigen

2. Transcription of a set of adenovirus early genes (the
E2 gene cluster) requires cell protein E2f (E2 factor)

- Now a family of proteins called the E2f family

3. E2f found to bind a cellular protein called
Retinoblastoma protein (Rb)

p53, Rb and E2F were subsequently discovered to be cri)cal players in control of the
normal cell cycle
 The cell cycle

G is for “gap” Resting cells are in “G zero”

When stimulated to divide, cells enter the G1 phase and then into S where they replicate
their DNA and prepare for cell division
Principles of Virology, ASM Press
 A go/no go decision is determined by
nutrient concentration and growth factors
Mitogenic signals

• Is the outside world rich enough to replicate the cell?

• Remember: Detectors and signaling proteins for
growth were discovered as oncogenes carried by
transforming retroviruses
Principles of Virology, ASM Press
 If conditions are not right, cell cycle
pauses at restriction point
Mitogenic signals

• No DNA synthesis, no cell

division

• The protein that regulates

the restriction point decision
is Rb Restric;on
point

ReMnoblastoma protein (Rb) – if both copies of the Rb gene are lost, develop reMnal tumors of
reMnoblasts that form reMna - these cells are gone by age 5

It is a recessive oncogene (the wild type protein is a tumor suppressor)

Principles of Virology, ASM Press
Principles of Virology, ASM Press
But DNA viruses need cells in S phase so
they can replicate their DNA

T antigens kick quiescent cells into S phase!!

When viral T antigens bind to Rb, E2f proteins
are released and initiate S phase transcription

(Ad transcription
also requires E2f)

Principles of Virology, ASM Press

The entry into S decision is under MORE control

• DNA damage or unscheduled DNA synthesis is monitored by p53

• Don’t duplicate damaged genetic information!

• Viruses must counter p53

Principles of Virology, ASM Press
How do viruses counter p53?

Principles of Virology, ASM Press

 Go to:

m.socrative.com
room number: virus

T antigens are:

1. Encoded by viral genes that are essential for replication

2. Present in tumors and transformed cells
3. Encoded by viral genes that have been incorporated
into the cell genome
4. Antagonists of cell cycle checkpoint proteins
5. All of the above

3
 Two more mysteries remain

1. Why are all viral genes EXCEPT T-antigen

encoding genes deleted or turned off in SV40,
polyma, and adenovirus- transformed cells?
2. Why is transformation by these viruses so
rare?
Transformation is rare because two low
probability events are required

1. Lethal late genes must not be expressed

- Rare spontaneous deletion of late gene
- Infection of semi-permissive cells; late gene expression
blocked

2. T antigen must be on constitutively and

transmitted to every cell
- Viral DNA encoding T antigen must be integrated into the
host DNA
 Transforma)on and tumor forma)on are abnormal,
epigene)c processes for these DNA “tumor” viruses

These events are not required for the normal viral life cycle
or transmission

We now understand that even in the natural host, these rare events may
occur, leading to tumorigenesis (e.g. HPV)
Transformation is an epiphenomena of a
unique reproductive cycle

• DNA tumor viruses must start the cell synthetic

machinery to make DNA

• T antigens turn on the cell cycle to start the G1

to S phases through inactivation of normal
inhibitor (Rb)

• Inactivation of p53 blocks apoptosis

If ly;c events are blocked, cells making T an;gens con;nue to divide – they are
transformed
They are on their way to becoming cancer cells
 Revealed by studying
transforming retroviruses
Dominant oncogenes

Revealed by studying
DNA tumor viruses
Recessive oncogenes

Principles of Virology, ASM Press