Genetic Alterations in Cancer Original slides by: Dr. B. F. Burns Dr. Bojana Djordjevic Department of Pathology and Laboratory Medicine.

Genetic Alterations in Cancer

Original slides by:

Dr. B. F. Burns

Dr. Bojana DjordjevicDepartment of Pathology and Laboratory Medicine

Objectives

• Discuss the common types of cancer-associated genes and provide examples of each, discussing their normal function and their effects when normal function is lost.

• Describe the common types of changes that can affect genes associated with cancer.

Genetic Alterations in Cancer• All neoplasms result from genetic changes

in the tumor cells• Changes may be:

• inherited - “cancer kindreds” (relatively rare)• mutations are in the “germ line”

• acquired (most common)• mutations are only in the tumor cells

Features common to cancer cells

1. Growth in the absence of “go” signals2. Growth despite “stop” signals issued

from neighboring cells3. Evasion of “auto-destruct” pathways in

response to genetic damage4. Stimulate local blood vessel growth5. Effectively immortal6. Locally invasive growth and metastases

Hanahan and Weinberg, Cell, 2000.

Basic Concepts in Oncogenesis

• Monoclonality• initial mutation occurs in a single cell

• mutated cell is effectively “immortalized”, either by replicating uncontrollably or not dying off normally

• replication of this cell results in “(mono)clonal expansion”

Basic Concepts in Oncogenesis

• Tumour progression• initial mutation is NOT sufficient to produce

a clinical “tumour”• genome appears to be unstable

(? DNA repair defect) leading to further mutations

• sequential mutations lead to subclones with progressively more “malignant” phenotypes (a nasty form of natural selection)

Hanahan and Weinberg, Cell, 2000.

Tumor cells begin as a clone, but become progressively more heterogeneous with time

TT

TN

T

T

T

T+

1. Initial mutation starts immortalized clone - not fully malignant yet

2. Second, third, etc. mutations give further growth advantages to sub-clones

T+

T

T

T*

T

T+

T+T+T+

T*

T*

T*T*

T*

T*T*

T TT

TT+

T+ T+T+

T+T+

T+

T+

T+

T+T+

T*

T*

3. Mature tumor contains many different sub-clonesAfter Robbin’s Pathologic Basis of Disease

Tumour cell kinetics• Tumour cells don’t always divide more

quickly than normal cells

• “Growth fraction” (proportion of cells actively dividing) usually increased

• tumour cells don’t die normally (growth by accumulation)

Classes of Genes involved in Cancer

• Cell cycle genes – control cell replication• Growth factors and receptors • Signal transduction proteins• Nuclear regulatory proteins• Tumor suppressor genes• Apoptosis (programmed cell death) proteins and

normal senescence (telomeres)• DNA repair genes

Cancer Associated Genes• All of these genes exist and function in normal cells as

1. proto-oncogenes

2. tumor suppressor genes

3. apoptosis genes.

• depending on their normal function they may contribute to a cancer by overactivity or underactivity and may behave in a dominant or recessive fashion in the cell.

• Overactivity may result from increased expression or decreased degradation (e.g.. mutations that prolong protein half-life)

Types of Genes associated with cancer

• “Classic” Oncogenes• promote cell growth and division• ras, myc and Her2/neu are examples

• Tumour Suppressor Genes• normally inhibit cell division• p53, Rb and BRCA-1 and -2 are examples

• Apoptosis Genes• associated with normal cell death and turnover• bcl-2 family is best known

“Classic” Oncogenes• Mutation or overexpression associated

with overactivity of gene

• genetically dominant effect

• example: ras point mutations are very common in human tumours• mutant ras signal transducer doesn’t need

growth factor binding to be active

Sites of action of “classic” oncogenes

• Growth factors

• Growth factor receptors

• Signal transduction proteins

• Nuclear regulatory proteins

• Cell cycle regulators

Mechanisms of Oncogene action - Growth Factor overexpression

Saturated receptors send excess growth signals

Overexpression of sis

Mechanisms of Oncogene action - Growth Factor Receptor overproductionHer2/neu overexpressed Excess receptors

make cells overly sensitive to existing growth factors

Her2 +

Mechanisms of Oncogene action – Signal Transducer Mutations

Mutant ras doesn’t need external activation

Signals activate transcription and cell division

Receptor on surface bypassed

Mechanisms of Oncogene Action -Mutated Nuclear Transcriptional Activators

Myc protein

Surface receptors bypassed

Myc proteinMyc protein

Myc protein

Myc protein

Myc protein

1. Mutated Transcriptional Activator gene

2. Excess myc production

3. Deregulated cell replication

Mechanisms of oncogene action - Cell cycle proteins

Cyclin D and the Cyclin-dependent kinases

G1

G2

S

Mcdk4

Cyclin D

Active complex controls G1 to S via Rb phosphorylation

•Overexpression of Cyclin D or cdk4 leads to loss of normal control of cell replication, occurs in many different tumors

Tumor Suppressor Genes - General features

• genes that normally function to suppress cell replication (not specifically to prevent tumors)

• can be considered to function in opposition to effects of “classic” oncogenes

• normal cell growth depends on a balance between the effects of proto-oncogenes and TSG’s

Tumour Suppressor Genes - Genetics

• Only need one good copy (wild type) of the gene to maintain function• “recessive” genetics at a cellular level• Theory may have to be modified slightly to recognize

“haplo-insufficiency”

• With one allele being mutated, a chance mutation of the second normal allele results in both functioning alleles being knocked out - referred to as “loss of heterozygosity”. The function of the TSG is lost and tumor develops. • TSG defects inherited in a dominant fashion

• ALL the family members who carry one mutated allele are at risk of cancer

Tumour Suppressor Genes - Genetics

• Tumors involving TSG• Familial

• Carry one mutated allele in the germline• Tumor arises when there is a sporadic mutation of the other

allele (loss of heterozygosity)• Early age of onset• Multifocal• Bilateral

• Sporadic• Both wild type alleles must undergo sporadic mutations in order

for tumor to develop• Later in life• Unifocal• Unilateral

Retinoblastoma (Rb): The “original” tumour

suppressor gene• First recognized TSG (1968), familial

inheritance of infantile eye tumours

• Knudson proposed that both alleles of the gene had to be mutated in order to see the effect (“two hit hypothesis”)

Clinical appearance of retinoblastoma - leukocoria

Normal “red eye”Leukocoria – white eye

Familial inheritance of Retinoblastoma

HeterozygousZygote:1 in 2 chance

Normalgene

Mutant Rb gene

Chance 2’nd mutation-virtual certainty

Homozygous mutant cell

Millions ofcell divisions

Retinoblastomaforms from thatcell

After Robbin’s Pathologic Basis of Disease

P53 - the prototype tumour suppressor gene

• p53 is the most common gene affected in human cancer

• normally functions as the “Guardian of the Genome” • mutagen exposure leads to increased T1/2 of p53, cell is

blocked in G1, allowing for DNA repair

• if repair fails, p53 induces apoptosis genes, cell dies

Genes affecting Apoptosis• another way in which tumours may form is

if a clone of cells fails to die in a normal fashion (apoptosis)

• the genes controlling this may either prevent apoptosis (e.g.. bcl-2 ) or induce it (e.g.. bax)

• within a cell the balance of these two determines whether the cell goes into apoptosis

How Genes affecting Apoptosis act

to produce tumors• bcl-2 protein functions to block programmed cell

death (apoptosis)• abnormal expression “immortalizes” the cell• such cells are at increased risk for additional

mutations of other oncogenes• t(14;18) translocation in follicular lymphomas

leads to increased bcl-2 expression

DNA-repair defects

• Not actually oncogenic proteins, but inability to repair ongoing mutations to other proto-oncogenes predisposes to cancer• Initially discovered via familial syndrome of

ataxia-telangiecatasia - ATM protein senses DNA damage, activating p53 cascade

• Mismatch repair protein defects in hereditary non-polyposis colon cancer

• Xeroderma pigmentosa patients cannot repair UV-light DNA damage

The Colon Cancer Sequence

Multistep mutations in Adenoma to Colon Cancer –

“Suppressor Pathway”Normal

Epithelium

APC gene lost on 5q

HyperplasticEpithelium

Ras mutationon 12

Low grade adenoma

Intermediate grade

adenoma

DCC losson 18

High grade adenoma

p53 loss on 17

Carcinoma

Metastases

? others

After Volgelstein & Fearon et alNEJM 1988;319:525-532

DNA methylationloss

Pathologic Correlates in theColon Cancer Sequence

Benign Tubular Adenoma of Colon

Colonic Carcinoma arisingin a pre-existing Adenoma

Adenoma

Carcinoma going through wall of colon

Stalk of normalcolon mucosa

Microscopic correlates in the Adenoma - Cancer sequence

High gradeDysplasia of Epithelium

NormalEpithelium

Low gradeDysplasia

Adenoma Carcinoma

Second mechanism of colon cancer development

• “Mutator pathway” – involved in 10-15% of all colon cancers and in all of the sub-group called “Hereditary non-polyposis colon cancer”

• Involves mutations to mismatch repair genes leading to “microsatellite instability” (MSI) in the genome

• Clinical importance is in: • pointing to family genetic testing for MSI• typically lower stage/better prognosis

Jass, Whitehall, et al. Gastroenterology 2002; 123: 862-876

Types of Genetic Changes affecting Cancer-associated genes

• Point Mutations- • activating proto-oncogenes e.g. ras • Inactivating tumor-suppressor genes, e.g. RB

• Aneuploidy - encompasses gross chromosomal changes with truncations, extensions or swapped segments such as:• Chromosomal Translocations-

• resulting in increased expression (bcl-2) or abnormal “fusion” protein (bcr-abl)

• Gene Amplifications-• increased copy number of genes, e.g. n-myc and

Her2/neu• Chromosomal deletions-

• esp. affecting tumor suppressor genes, e.g. Rb deletions at 13q14

Chromosomal Translocations

• Most common in leukemias and lymphomas

• Many are characteristic of specific types:• t(9;22) translocation in chronic

myelogenous leukemia

• t(8;14) in Burkitt lymphoma

• t(14;18) in follicular lymphoma

Chromosomal Translocations

From Robbin’s Pathologic Basis of Disease, 5’th Ed.

Chromosomal Translocations

From Robbin’s Pathologic Basis of Disease, 5’th Ed.

Gene Amplification

• An increase in the copy number of certain oncogenes leads to over-expression

• Her2/neu most common example

• n-myc amplification is common in pediatric neuroblastomas • larger copy number is associated with

worsening prognosis

Summary points:• Genetic changes are at the root of all cancer• A given type of cancer may arise and evolve

genetically in an extremely variable fashion• Within a single tumor the genetic abnormalities

are variable from cell to cell

Further reading

• Robbins and Cotran Pathologic Basis of Disease. 9’th Ed. 2015. Pages 282-306.• This is the portion of the Neoplasia chapter

dealing with genetic alterations and cancer-associated genes.