From tumour viruses to

oncogenes

Week 2: Lecture 2

Michael Olson
BMS850_W2023
 Cancer has aberrant
chromosomes

Sources:
Robert Weinberg: The Biology of Cancer – Chapter 2
Lewis J. Kleinsmith: Principles of Cancer Biology
CANCER

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 Chromosomes are aberrant in cancer cells
• PIONEERING THEORY

Theodor Boveri (1862-1915)

“Tumour growth is based on

a particular, incorrect
chromosome combination
which is the cause of the
abnormal growth
characteristics passed on to
daughter cells”

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“Concerning the Origin of Malignant Tumours”
 Chromosomes are aberrant in cancer cells
• PIONEERING TECHNIQUE
– CYTOGENETICS = study of number and structure of chromosomes

Giemsa (G) chromosome banding technique

Metaphase

Staining Banding

Karyotype (number and appearance) of human chromosomes

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 Chromosomes are aberrant in cancer cells
• PIONEERING TECHNIQUE
– CYTOGENETICS = study of number and structure of chromosomes

Chromosome mapping

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 Chromosomes are aberrant in cancer cells
• PIONEERING TECHNIQUE
– CYTOGENETICS = study of number and structure of chromosomes

Spectral karyotyping (SKY) – fluorescence painting of chromosomes

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 Chromosomes are aberrant in cancer cells
• The first experimental evidence of
chromosomal aberrations in Philadelphia chromosome in CML
human cancer
• Abnormally configured
chromosome in a large
proportion of CML patients

CML=chronic myelogenous leukemia

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 Chromosomes are aberrant in cancer cells
• Chromosomal rearrangements in cancer

Philadelphia chromosome in CML

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CML=chronic myelogenous leukemia
 Chromosomes are aberrant in cancer cells
• Chromosomal imbalances in cancer (gains and loses)
Normal
Cancer

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 Chromosomes are aberrant in cancer cells

• Chromosomal rearrangements
– Translocations = rearrangement of parts between nonhomologous
chromosomes
– Inversions = segment of a chromosome is reversed end to end

• Chromosomal imbalances
– Genomic gains
– Genomic losses

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CANCER

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 Cancer genes

• Oncogenes and Tumour Suppressor genes

Oncogenes = promote cancer

Tumour suppressor genes (TSG)s = protect from cancer

Protein expression
Cancer gene Normal cell Cancer cell
Oncogenes LOW HIGH
Tumour suppressor genes HIGH LOW

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BOTH are misregulated in cancer

TSG ONCOGENE

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 Discovery of oncogenes

Sources:
Robert Weinberg: The Biology of Cancer – Chapter 3
Lewis J. Kleinsmith: Principles of Cancer Biology
 Discovery of oncogenes

• Tumour viruses

Payton Rous – Discovery of Rous Sarcoma Virus (RSV) - 1910 16

Key finding 1:
Rous induced sarcomas in chickens

Transplantation experiments

• Tumours can be excised from tissue from one

animal and forced to grow as a graft in the body of
another animal (same or different species)
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 Rous induced sarcomas in chickens

Rous Sarcoma Virus

• RSV filtrate caused sarcoma

• RSV filtrate is transmissible

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 Rous induced sarcomas in chickens

Rous Sarcoma Virus

• https://www.pbs.org/video/story-cancer-emperor-all-maladies-
discovery-oncogene-carcinogeneis/

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Key finding 2:
In vitro transformation of normal cells to tumour
cells after RSV infection

• contact inhibition – proliferation stops upon cell contact

– cancer cells lose contact inhibition and continue proliferation
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Contact inhibition

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 HALLMARKS OF
TRANSFORMED CELLS

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EXPERIMENTAL ASSAYS
FOR CELL
TRANSFORMATION

Foci forming assay

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EXPERIMENTAL ASSAYS
FOR CELL
TRANSFORMATION

Soft agar assay

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 EXPERIMENTAL ASSAYS
FOR CELL
TRANSFORMATION

Tumorigenicity assay

Ability of transformed cells to form tumours in mice

Subcutaneous injections = under skin

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Orthotopic injections = organ specific Nude immunocompromised mice
 Notes on tumorigenicity assay
• Immune system eliminates foreign tissue
– Barrier to transplantation
• Immunocompromised strains of mice
– Lack fully functional immune systems
– Tolerate engrafted tissues of foreign genetic origin including
xenografts (transplanted tissues from other species)
• Example strains:
– NUDE – absent thymus – reduced T lymphocytes
– SCID – severe combined immunodeficiency
• Absent T and B lymphocytes

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Key finding 3:
Continued presence of RSV is needed to
maintain transformation

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 Lessons learned from RSV…

• RSV transforms normal fibroblast cells to highly

proliferative, anchorage-independent and tumorigenic
cells

• Development of assays for experimental tumorigenesis

• RSV virus is required to initiate and maintain the

transformed phenotype of fibroblast cells

• RSV can be passed from a transformed mother cell to its

daughter cells

HOW?
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Key finding 4:
Re-infection could not explain the stable
transmission of RSV genome

Wild type RSV

Replication-deficient RSV

Re-infects daughter cells

RSV left something behind
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RSV is a retrovirus

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The life cycle of an RNA virus

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Key finding 5:
v-src gene is required for transformation
core RT spikes

Transformation
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Are src sequences integrated in the genome of the infected RSV cells?
 Detecting src gene in host genome DNA
• Southern blotting

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Key finding 6:
src gene is present in uninfected cells

src sequences were found

in both RSV infected and
uninfected cells

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Key finding 7:

src gene is normal, highly conserved gene

of all vertebrate species

src sequences were

found in other species

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Kidnapping of src

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 Proto-oncogenes
• cellular src or c-src is present in normal tissues
• c-src is not cancer causing
• c-src has a potential to transform cells under certain circumstances (if
activated as in v-src)
• c-src is a proto-oncogene

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The vertebrate genome carries a large group
of proto-oncogenes

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 Insertional mutagenesis

Some retroviruses do not

kidnap cellular proto-
oncogenes but can transform
ALV
cells by integrating in the
DNA and acting like
transcriptional promoters.

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Cellular oncogenes activated by insertional
mutagenesis

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 Key points

• Many carcinogens act by mutating DNA

• Mutagens are identified using AMES test
• Mutations and chromosomal abnormalities are
present in cancer cells
• Genes involved in cancer are known as
oncogenes or tumor suppressor genes
• Oncogenes were discovered in retroviruses
• Proto-oncogenes are cellular genes that have
the ability to become cancer causing

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 Cellular oncogenes

Sources:
Robert Weinberg: The Biology of Cancer – Chapter 4
Lewis J. Kleinsmith: Principles of Cancer Biology
 Discovery of human oncogenes
• Research question: Does bladder carcinoma DNA carry
cancer causing genes (oncogenes)?

TUMOUR TISSUE

Bladder carcinoma

• Transformation occurs at low frequency

– Only a small subset of genes are causing transformation 43
 MOUSE FIBROBLAST CELLS

Focus generated UNTRANSFORMED:

TRANSFORMED:
by transfection of - Wide cytoplasm
- Refractile
DNA from the human - Single monolayer
- Densely packed
bladder carcinoma - Do not pile up
- Spindle shape

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Are oncogenes discovered in human tumour cell
lines related to those carried by transforming
viruses?

DNA isolation Retroviral oncogene

from foci cells DNA probes

Southern blotting

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Oncogenes discovered in human tumour cell
lines are related to those carried by
transforming retroviruses

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Weinberg discovered mutant version
of a proto-oncogene ras

Weinberg’s oncogene was related to the genome of Kirsten Sarcoma Virus (KRAS)

What is the consequence of this mutation? 47

Weinberg discovered mutant version
of a proto-oncogene ras

https://www.pbs.org/video/story-cancer-emperor-all-maladies-dr-robert-
weinberg/)

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 Constitutive activation of ras

Normal cell Cancer cell

Blockage caused by
oncogenic mutation

Regulates cell proliferation and movement 49

 Constitutive activation of ras

• RAS is a GTP binding protein that normally cycles between an

active GTP bound state and inactive GDP state

• ON and OFF state is regulated through GTP hydrolysis

• Ras point mutation affects the GTP hydrolysis site inhibiting

GTP hydrolysis

• Ras protein gets stuck in its GTP bound state

• Constitutive activation – always ON

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 Kras Structure

• http://www.rcsb.org/3d-view/4OBE/1
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Kras Structure

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 Ras Isoforms are
frequently mutated in
human cancers
Ras subfamily:
- Family of related proteins expressed in
all animal cell lineages and organs.
- Small GTPase proteins involved in
transmitting growth signals in cells
Ras subfamily:
- H-ras (Harvey ras)
- K-ras (Kristen ras)
- N-ras (Neuroblastoma ras)
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 Mechanisms of oncogene activation

• Gain-of-function mutations (e.g. ras)

– Point mutations in a proto-oncogene that result in a
constitutively acting protein product

• Gene amplification
– Increase in copy number of a gene within the
genome of a cell

• Chromosomal Rearrangements
– Chromosomal translocations and chromosomal
inversions

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 Gene amplification

• Consequences of gene amplification

Oncogene
Increase in gene copy number

How do we detect these changes in patient samples? 55

 Detecting gene amplifications in cancer

• DNA copy number amplification detection

– FLUORESCENT IN SITU HYBRIDIZATION (FISH)

• Protein overexpression detection

– IMMUNOHISTOCHEMISTRY

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 Fluorescent in situ hybridization (FISH)

• Cytogenetic technique that uses fluorescent probes that bind to

only those parts of the chromosome with a high degree of
sequence complementarity.

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 HER2 amplification in breast cancer
HER2 negative HER2 amplified

Red dot: HER2 copies

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Green dot: Centromere protein copies (Control)
HER2 protein overexpression in breast cancer

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Her2 oncogene amplification in breast cancer

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N-myc amplification in childhood
neuroblastomas

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 Mechanisms of oncogene activation

• Gain-of-function mutations (e.g. ras)

– Point mutations in a proto-oncogene that result in a
constitutively acting protein product

• Gene amplification
– Increase in copy number of a gene within the
genome of a cell

• Chromosomal Rearrangements
– Chromosomal translocations and chromosomal
inversions

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 Chromosomal rearrangements

• Type of chromosomal rearrangements that activate proto-oncogenes

– The creation of fusion genes (common in hematopoietic cancers)
• bcr-abl oncogene in leukemia
– The transcriptional activation of proto-oncogenes
• c-myc in Burkitt’s lymphoma

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 Creation of fusion genes

• P120 bcr-abl oncogene in CML (Philadelphia chromosome)

BCR-ABL=oncogene

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Transcriptional rearrangements

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 Transcriptional rearrangements

Myc is a transcription factor – promotes transcription

Normal Myc protein is expressed but in abnormal amounts!

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CONSTITUTIVE EXPRESSION OF MYC through transcriptional activation by IgH enhancer sequences
 Many oncogenes can be activated via different
mechanisms

• Myc
– Gene amplification (neuroblastoma)
– Chromosomal translocation (Burkitt’s lymphoma)
• Kras
– Point mutations (pancreas, lung, colorectal)
– Amplifications (pancreas)
Homework: Can you find more examples?

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 Key points

• The first human oncogene Kras was discovered

using gene transfer techniques.
• Oncogenes are activated via three main
mechanisms:
– Point mutations, amplifications, translocations
• FISH and IHC techniques are used clinically to
determine the amplification of certain
oncogenes
• Same oncogene can be activated via different
mechanisms
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