Tumour Suppressor Genes CLS 712 2022-2023

Tumour Suppressor Genes
TSG
Advanced Immunology I
CLS 712
January 4th 2023
Course Coordinator :
Dr. Lamya Zohair Yamani
*The Biology of Cancer
Weinberg
Prepared by Dr. Lamya Yamani Postgraduate (MSc) CLS 712 2022-2023
The Biology of Cancer Robert A. Weinberg
“ Another possibility is that in every normal cell there is a specific arrangement for inhibiting
growth; which allows the process of division only when inhibition has been overcome by
special stimulus.
The presence of definitive chromosomes which inhibit division would harmonize best with
fundamental ideas.....
That cells of tumours with unlimited growth would arise if those “inhibiting chromosomes”
were eliminated”.
Theodor Boveri 1911

Experimental Fusion of Cells
❑ Familial cancer studies suggested

cancer was a dominant trait
❑ Studies utilizing somatic hybridization

suggested the opposite
▪ Cell fusion to create hybridomas, used

Sendai virus or polyethylene glycol
(PEG) to fuse cell membranes
together. Special drug selection
schemes allowed selective
maintenance of the hybrid cells only.

Heterokaryon Polykaryon

Dominance or Recessiveness of the Tumourigenic
Phenotype?

Cancer Cellular Phenotype More likely to be Recessive
• A recessive cancer phenotype was found to be a general phenomenon
• True for cancer cell lines from tumours induced by various means (chemical, irradiation, or viruses)
• Reversion of non-tumourigenic hybrid to tumourigenic state was observed in some circumstances
• Associated with loss of specific normal chromosomes or chromosomal regions
• This was the clue for Tumour Suppressor Gene existence.

Familial Involvement Percentages of all Cancers
Cancer May Cluster in FamiliesExamples
None (sporadic) More than 90% All types
Involved 5-10 % Colon, Breast
Well defined 0.1 % Familial Retinoblastoma
Involved: Some evidence for hereditary predisposition
Well defined: Cancer inherited with a high prevalence in a pedigree (very rare)

Family History “Involved” in Prostate Cancer Risk
• Prostate Cancer 29% of male cancers are prostate cancers
• Familial aggregation for prostate cancer is recognized in about 20% of prostate cancers

Pedigree Typical of Familial Retinoblastoma “Well
defined” Inherited Predisposition
Image taken from google images

Pediatric Retinoblastomas
Develop from retinoblasts in the

embryionic eye precursors to multiple cell
types present in the retina

Pediatric Retinoblastomas

Unilateral Vs. Bilateral Retinoblastomas
A scientists by the
name of Knutson
realized that
inherited
retinoblastoma was
frequently bilateral
(meaning both eyes
have tumour)
whereas sporadic
forms are usually
unilateral (one eye
has tumour)

Patients with
bilateral
retinoblastomas
also:
• Are prone to
tumours later in
life
• Various tissues
but especially
sarcomas

Kinetics of Appearance of Unilateral and Bilateral
Retinoblastomas
The frequency and

incidence versus
age of sporadic
retinoblastoma fit
model of 2
independent
equally frequent
events occurring in
the same cell.

Dynamics of Retinoblastoma Formation

Chromosomal Localization of the Locus
Clues were
obtained from
cytogenetic
techniques;
Retinoblastoma
cells had specific
deletions

Loss of Heterozygosity (LOH), Studies using Molecular
Markers
This shows loss of

heterozygosity at
the Rb locus

Restriction Fragment Length Polymorphism
(RFLPs)
• Used for
distinguishing
whether DNA
from both
homologs are
present in a
tumour
genomic DNA
sample
• This helped to
localize the
Tumour
suppressor gene

Elimination of WT
Rb gene copies

How were these hypothetical TSGs actually identified?
• If familial cancer is the result of inheritance of some cancer causing allele of a gene
then it should be possible to establish linkage between known genetic markers and
the cancer gene. This is the first step to positional cloning of the TSG
Going from linkage to cloning TSG:
• A genetic linkage map would show the relative locations of specific DNA markers or
loci along the chromosome
• Any inherited physical or molecular characteristic that differs among individuals and is
easily detectable in the laboratory is a potential genetic marker or locus.
• Markers must be polymorphic to be used for mapping, alternative forms must exist
among individuals so that they are detectable among different members in familial
studies.

What are the steps taken to confirm and study TSG?
Are structural defects present in both alleles of TSG in the tumour cells?
Do normal cells from the patient of familial cases harbor one or two normal alleles from
normal cells?
Is the TSG protein absent or does it lack critical biochemical activity in the tumour cells?
Does reintroduction of a cloned WT TSG into the TSG negative tumour cell line result in
growth suppression while cloned mutant TSG show no effect?
What is the phenotype of mice made to carry one mutant allele of the TSG ? Or homozygous
mutant for the TSG?

Loss of Heterozygosity of Chromosomal Arms in Colon
Cancer
• Occurs on many
chromosomes
• May be many
unidentified
TSGs

• In tumour DNA LOH is observed when chromosomal loss with or without chromosomal
reduplication occurs, when homologous recombination occurs or large deletions occur.
• Quantitation of the probe signals and examination of polymorphic markers all along the
chromosome can distinguish these mechanisms.
• Observing LOH for markers in a given chromosomal region in a multiple tumour suggests
the presence of TSG

• Maintenance of
DNA
methylation
following
replication
• In contrast to
most CpG
dinucleotides
those near
active
promoters are
non methylated
• Methylation of
these promoter
CpGs silences
promoter
Example:
Methylation of the
promoter RASSF1A

Methylation of
multiple genes
within the tumour
cell genome

NF1 and RAS Signaling
Neurofibromatosis Type I (NF1) Syndrome:
• An autosomal dominant cancer predisposition
• Observed in 1: 3500 people
• Signs: Cafe au lait spots, neurofibromas, Lisch nodules
• Various benign tumours are seen in high frequency , subcutaneous

neurofibromas, and optic gliomas.
• Has increased incidence of various malignant tumours acute and chronic

forms of childhood myeloid leukemias, neurofibrosarcomas and glial cell
tumours
• Patients of NF1 have inherited one mutant copy of the NF1 gene and the
tumours that appear have undergone somatic loss of the WT NF1 allele.
Neurofibromatosis

NF1+ / NF1+ NF1+ / NF1-
NF1+ / NF1-
Image adapted

Neurofibromin:
• Expressed ubiquitously
• Highest expression found in neurons, Schwann cells,

oligodendrocytes
• Over 50 exons spread throughout 300kb on chromosome 17
• Multiple mutations found in exons 11-17, 20-27, and 37

NF1 Negatively Regulates RAS
Proliferation
Differentiation
Survival
Image taken from google images

Neurofibromin and RAS Signaling Pathway
• Increased RasGTP levels compared to

normal cells
• This effect has been shown to be

pronounced in response to specific
growth factors
• NF1- negative cells are hypersensitive

to the growth promoting effects of
these same growth factors

PTEN
• A variety of tumours have acquired deletions involving the 10q23-24 region or show LOH for
markers in this region. For example 90% of glioblastomas have one copy of chromosome 10
partially or completely deleted. Therefore a TSG was suspected to reside on 10q23-24.
• Identified in 1997 using positional cloning approaches or as a TGF-β downregulated gene, in

different labs hence the multiple names
PTEN; Phosphatase and tensin homolog deleted on chromosome ten
MMAC1; Mutated in multiple advanced cancers 1
TEP1; TGF β regulated and epithelial enriched phosphatase 1

Continuation:
• PTEN is a phosphatase (needed to dephosphorylate substrates)
• PTEN shows biallelic inactivation in many spontaneous cancers;
~ 30% of endometrial cancers

~30% prostate cancers
~20% lung cancers
~10% breast cancers
~15% melanomas
• PTEN is inherited in mutant form in certain autosomal dominant cancer predisposition

syndromes ~80% Cowden disease, Bannayan-Zonana syndrome
• PTEN antogonizes signaling via the PI3K. Therefore when PTEN is lost from tumour cells PI3K
signaling is hyperactive and the balance between cell survival and death is shifted towards
survival.

Images taken from google images


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Tumour Suppressor Genes

Theodor Boveri 1911

Prepared by Dr. Lamya Yamani Postgraduate (MSc) CLS 712 2022-2023

❑ Familial cancer studies suggested

❑ Studies utilizing somatic hybridization

▪ Cell fusion to create hybridomas, used

Prepared by Dr. Lamya Yamani Postgraduate (MSc) CLS 712 2022-2023

Prepared by Dr. Lamya Yamani Postgraduate (MSc) CLS 712 2022-2023

Prepared by Dr. Lamya Yamani Postgraduate (MSc) CLS 712 2022-2023

• A recessive cancer phenotype was found to be a general phenomenon

• Reversion of non-tumourigenic hybrid to tumourigenic state was observed in some circumstances

• Associated with loss of specific normal chromosomes or chromosomal regions

• This was the clue for Tumour Suppressor Gene existence.

Prepared by Dr. Lamya Yamani Postgraduate (MSc) CLS 712 2022-2023

Involved 5-10 % Colon, Breast

Well defined 0.1 % Familial Retinoblastoma

Involved: Some evidence for hereditary predisposition

Prepared by Dr. Lamya Yamani Postgraduate (MSc) CLS 712 2022-2023

• Prostate Cancer 29% of male cancers are prostate cancers

Prepared by Dr. Lamya Yamani Postgraduate (MSc) CLS 712 2022-2023

Image taken from google images

Prepared by Dr. Lamya Yamani Postgraduate (MSc) CLS 712 2022-2023

Develop from retinoblasts in the

Prepared by Dr. Lamya Yamani Postgraduate (MSc) CLS 712 2022-2023

Prepared by Dr. Lamya Yamani Postgraduate (MSc) CLS 712 2022-2023

Prepared by Dr. Lamya Yamani Postgraduate (MSc) CLS 712 2022-2023

Prepared by Dr. Lamya Yamani Postgraduate (MSc) CLS 712 2022-2023

The frequency and

Prepared by Dr. Lamya Yamani Postgraduate (MSc) CLS 712 2022-2023

Prepared by Dr. Lamya Yamani Postgraduate (MSc) CLS 712 2022-2023

Prepared by Dr. Lamya Yamani Postgraduate (MSc) CLS 712 2022-2023

This shows loss of

Prepared by Dr. Lamya Yamani Postgraduate (MSc) CLS 712 2022-2023

Prepared by Dr. Lamya Yamani Postgraduate (MSc) CLS 712 2022-2023

Prepared by Dr. Lamya Yamani Postgraduate (MSc) CLS 712 2022-2023

Going from linkage to cloning TSG:

Prepared by Dr. Lamya Yamani Postgraduate (MSc) CLS 712 2022-2023

Prepared by Dr. Lamya Yamani Postgraduate (MSc) CLS 712 2022-2023

Prepared by Dr. Lamya Yamani Postgraduate (MSc) CLS 712 2022-2023

Prepared by Dr. Lamya Yamani Postgraduate (MSc) CLS 712 2022-2023

Prepared by Dr. Lamya Yamani Postgraduate (MSc) CLS 712 2022-2023

Prepared by Dr. Lamya Yamani Postgraduate (MSc) CLS 712 2022-2023

• An autosomal dominant cancer predisposition

• Observed in 1: 3500 people

• Signs: Cafe au lait spots, neurofibromas, Lisch nodules

• Various benign tumours are seen in high frequency , subcutaneous

• Has increased incidence of various malignant tumours acute and chronic

Prepared by Dr. Lamya Yamani Postgraduate (MSc) CLS 712 2022-2023

Prepared by Dr. Lamya Yamani Postgraduate (MSc) CLS 712 2022-2023

• Highest expression found in neurons, Schwann cells,

• Over 50 exons spread throughout 300kb on chromosome 17

• Multiple mutations found in exons 11-17, 20-27, and 37

Prepared by Dr. Lamya Yamani Postgraduate (MSc) CLS 712 2022-2023

Image taken from google images

Prepared by Dr. Lamya Yamani Postgraduate (MSc) CLS 712 2022-2023

• Increased RasGTP levels compared to

• This effect has been shown to be

• NF1- negative cells are hypersensitive

Prepared by Dr. Lamya Yamani Postgraduate (MSc) CLS 712 2022-2023

• Identified in 1997 using positional cloning approaches or as a TGF-β downregulated gene, in

PTEN; Phosphatase and tensin homolog deleted on chromosome ten

MMAC1; Mutated in multiple advanced cancers 1

TEP1; TGF β regulated and epithelial enriched phosphatase 1