Abnormalities of growths and tumours Flashcards
Abnormalities of growth and differentiation can result in
Fetal death (miscarriage)
Fetal abnormalities
-Anatomical defects - cleft palate, neural tube defects, atrial septal defects
-Biochemical/functional defects
-> Phenylalanine hydroxylase deficiency - leads to mental retardation due to accumulation of phenylalanine
Defective cell membrane transport (CFTR) gene in secretory genes - cystic fibrosis
Abnormal haemoglobin formation - results in sickle cell anaemia
Rare childhood syndromes
Progenitor cells
Division of stem cells also produces a daughter progenitor cell. Amplification of the progenitor cells allows growth.
Mitosis max 50-70 times
Labile cells
Cells that divide rapidly and complete a cycle every 16-24h
Stable cells
Resting or quiescent
They are in G0 of the cell cycle & can divide when stimulated
Permanent cells (static)
do not typically divide in adult life
Labile cell examples
Barrier tissues & the haematopoietic system are in a constant
state of renewal to replace lost cells.
- Many cells in these tissues will be labile cells (in constant cell cycle)
Rapidly proliferating progenitor cells are sensitive to…
insult - radiation, toxic chemicals/mutagens and are at increased risk of random damage to the genome
Stable cell example
Parenchymal tissue of many organs - hepatocytes, renal tubular epithelium
Static (permanent cells) example
Often the case for mature highly differentiated complex cells “terminally differentiated”
e.g cardiac myocytes or neurons
Hypertrophy
increase cell size (volume)
In static highly differentiated complex “permanent” cells
hypertrophy is often the only adaptive option for increased
functional demand
– cannot easily increase cell number by replication
e.g skeletal muscle
Hypertrophy disease
Cardiac hypertrophy
The number of myocardial muscle fibres does not typically increase (they are comprised of static “permanent” cells)- but their individual size (volume) can increase in response to an increased requirement. This leads to a thickening of the left ventricle
Hyperplasia
Increased cell number
Hyperplasia may be achieved by both
– An increase in cell number due to replication
– And/or a decrease in cell loss by apoptosis
- E.g. Hyperplasia of the erythropoietic system
- Increased numbers of erythrocytes (RBC) at altitude due to increased levels of
erythropoietin - Hyperplasia combined with hypertrophy also occurs
- Enlargement of sex organs at puberty
- Enlargement of breast tissue in pregnancy
– Due to the influence of increased oestrogen and testosterone etc
Hyperplasia disease
Endometrial hyperplasia
An increase in the
number of glandular
endometrial cells
- Due to high levels of
oestrogens, combined with
insufficient levels of the
progesterone-like hormones
in conditions such as
polycystic ovary syndrome
Adaptive atrophy
Atrophy results in a decreased size of the organ
* This may be due to a decrease in cell size and/or cell number
* This results in a diminished functional ability
- This adaptive response is due to a decreased requirement for
function of the cell/tissue - Importantly this this adaptive response can be reversible
- If due to a decrease in cell size
- or if due to a decrease in cell number in a labile or stable tissue
- In contrast cell death in tissues which cannot typically replace lost static
(permanent) cells this type of loss results in irreversible cellular atrophy
Disuse atrophy
Decreased function of a limb due to fracture
Immobilisation or loss of innervation of muscle
- Paralysed limbs in poliomyelitis - muscle atrophy
Nutritional atrophy
Starvation
Arterial disease (inferes with blood supply)
- loss of fat and muscle tissue
Physiological atrophy
Normal aspect of aging
- decrease in endometrial cellularity after menopause
Skeletal muscle atrophy
The number of muscle fibres (fused myocytes = mytotube fibre) is the same as before the atrophy occurred, but the size of some fibres is decreased
Hypoplasia
Failure of a tissue to reach normal size during development
– There is decreased proliferation compared with normal
* Or a mismatch between replacement and death of cells
– “underdeveloped”
- E.g. Achondroplasia
– an autosomal dominant mutation in the fibroblast growth factor receptor gene 3 (FGFR3) causes abnormal Impaired growth of cartilage
Differentiation is controlled by
Controlled by selective transcription of genes
– Inherited genome
- Maintained through interactions with other
cells
– E.g. growth factors/cytokines produced by cells,
the extracellular matrix secreted by the cell
(communication) - Influenced by
– Environmental (acquired) factors
Metaplasia
Changes in environmental/cellular signals can lead to an acquired change in differentiation of a cell this is called metaplasia
- It is the result of a gene–environment
interaction - It is an adaptive and reversible process
- Results in a different differentiation state more suited to the environmental insult/stimulus
Types of metaplasia
- Squamous metaplasia
– Change a complex functional type to a simpler cell type - E.g. Columnar epithelium -> squamous epithelium
- Glandular metaplasia
– change into a cell type with a more complex function
– Squamous epithelium -> glandular epithelium
Squamous metaplasia
Columnar epithelial cells change into squamous epithelium
– differentiated cells which were committed to a
specialised function (e.g. mucus secretion) change to a
simpler form
- E.g. Ciliated respiratory epithelium of the trachea and
bronchi in smokers
– ciliated columnar mucin-secreting cells of the bronchial
epithelium changed into stratified squamous epithelium with keratinization
Squamous metaplasia of respiratory epithelium
The chronic irritation of cigarette smoke has led to a change of of the normal columnar respiratory epithelium into more resilient
squamous epithelium
Glandular metaplasia
Glandular metaplasia is a change of cell type into a
more complex glandular type
- E.g. Transformation of the normal squamous epithelia
of the oesophagus to a gastric-type epithelium
resembling the stomach/intestinal mucosa - Due to chronic gastro-esophageal reflux disease (GERD)
– The metaplastic mucosa is better adapted and protected
against gastric acid than the squamous epithelium
Metaplasia and risk of dysplasia
Cells undergoing chronic insult undergo metaplasia (to change
into a more “resistant” cell type)
* The chronic damage may still lead to cell injury and loss of cells
- These cells are replaced by the proliferation of the progenitor cells in that tissue
- The progenitor cells undergoing mitosis are at increased risk of somatic mutations
– particularly due to mutagens from the source of the environmental insult - This accumulation of mutations in the somatic genome increases the chance of dysplasia (abnormality)
Abnormal organisation of cells in the tissue plus abnormal differentiation of cells results in
dysplasia
lack of ordered structure of aberrant cells
Abnormal differentiation & cellular communication
There is still transcription of “selected” genes of cell genome but….
– some of these genes may now contain somatic mutations
– some of these mutations will be in genes involved in the transcriptional regulation of the genome
* There may be atypical expression of genes which would normally be “off”
- The somatic mutations will
– Lead to errors in the genome – observed as increased nuclear size & chromatin content
– Adversely affect the normal “communication” between cells and the normal maintenance of an ordered structure within a tissue will be lost
– Lead to errors in the normal differentiation state of the cell- observed as atypical cells with unusual shape and size
Dysplasia and cell growth
There will be mitotic proliferation of cells in this
tissue to replace dead/damaged cells
- There will be an increased growth rate (increased
number of labile cells) in this dysplastic tissue - BUT these cells still can only replicate a finite
number of times
Dysplasia features
- Increased (normal) replication rate in response to cellular
injury - Increased number of mitotic cells (labile cells) in the tissue
- Aberrant cellular communication & atypical tissue
architecture - Accumulation of abnormalities (mutations) in the somatic
genome - Atypical cells (abnormal differentiation) with atypical nuclei
Dysplasia condition
Dysplasia is a pre-neoplastic condition
- Mild/early forms of dysplasia may reverse if the chronic
stimulus is removed - Severe dysplasia can progress to development of neoplasm
– This requires additional (cumulative) somatic mutations which lead to
the acquisition of autonomous (immortal) proliferation of cells - Dysplasia may be present for many years prior to progression to
neoplasia
– Screening for dysplastic lesions may be useful to prevent
severe malignant disease E.g. Cervical smear test
Neoplasia
Dysplasia PLUS the acquisition of autonomous
(immortal) proliferation of cells
* “new” growth
* these cells can replicate indefinitely
- This is the result of additional (cumulative) somatic mutations
within the dysplastic tissue - The resulting tissue is called a neoplasm
Abnormal (aberrant differentiation) mass of cells with excessive autonomous growth uncoordinated with
surrounding normal tissue
Neoplasms
Neoplasms result from uncontrolled excessive
autonomous growth and the disordered (aberrant)
differentiation & organisation
– Neoplasms may only stop growing when there is no more
oxygen and nutrients
* death of the patient
Immortality
Normal cells can only replicate a limited number of times (50-
70 times)
– telomeres located at the ends of chromosomes, get slightly shorter with each new cell division until they shorten to a critical length and become uncapped
- This is known as replicative senescence (Hayflick limit)
– i.e. growth arrest, cannot divide- therefore cannot replace lost cells - In contrast:- Neoplastic cells are “immortal”
– They express the ribonucleoprotein telomerase
– Telomerase adds new DNA onto the ends of the chromosome
– This maintains stable telomere ends to the chromosome
– This allows the cell to replicate indefinitely
Neoplastic cell immortality
Telomerase synthesises new DNA to lengthen 3’ overhang
Infinite number of mitoses= immortal
Neoplastic cells “switch on” the gene that leads to expression of the telomerase enzyme
Can always form a
telomere regardless of
the number of cell
divisions
Growth (excessive proliferation)
Neoplastic cells and cell cycle
Can overcome normal cell cycle checkpoint.
Proteins at G1 (restriction point)
Key proteins at this checkpoint are cyclin D4 and pRB
Somatic mutations forming a neoplastic cell
The change from a normal to a neoplastic cell is due
to a mutational event in the cell genome (somatic
mutation)
- Mutation is the result of
– spontaneous errors during cell division
– DNA damaging chemical, physical, or biological agents - These changes in the cell genome lead to:
– Activation of oncogenes
– Loss of tumour suppressor genes - Most neoplasms arise from the clonal expansion of a single cell that has
undergone neoplastic transformation
– The daughter cells ‘inherit’ the somatic mutation
In neoplasia multiple genetic mutations cause
abnormal (inappropriate) differentiation
aberrant lack of control (dysfunctional cellular
communication & atypical tissue architecture)
autonomous & immortal cell growth (proliferation)
Abnormal relationship with stroma (neoplastic cells)
– Lack of appropriate cell-to-cell communication
* Uncontrolled growth
– Recruit the surrounding tissue to provide nutrients/O2
* Angiogenesis
– Invade the surrounding tissue
* Epithelial- mesenchymal transition
* motile
(malignant)
Benign
Grow as a compact mass (within) the tissue as they expand
* Distort but don’t destroy and invade
Malignant
Cells invade and destroy the surrounding tissue as they
grow and may spread to distant sites
Benign neoplasia
Benign tumours do not metastasise
– Confined to site of origin
* Benign tumours are expansive
– Benign epithelial neoplasms often form “polyps”
- Histologically closely resemble the parent cell
- Growth rate is relatively slow
- There is an abnormal relationship to
surrounding tissue
– Lack of appropriate cell-to-cell communication
– Angiogenesis to provide nutrients/O2
Benign can cause
Cause pressure atrophy of
the surrounding
parenchymal tissue
– Obstruction of flow of fluid
(benign tumour of a duct)
– Benign meningeal tumour
causing epilepsy
- Inappropriate effects, such
as excessive production of
hormone e.g. benign thyroid
tumour
Malignant
Invasive
- Grow in an irregular pattern into the surrounding tissue
- Destroy the surrounding tissue as they invade
- Spread (metastasis)
- neoplastic cells access the lymphatic or blood vessels and are then carried to
distant site to form a new malignant growth - malignant neoplasia = CANCER
- cancer causes considerable mortality and morbidity
- destruction of adjacent tissue
- formation of secondary tumours
- blood loss from ulcerated surfaces
- pain
- obstruction of flow in ducts/vessels
- inappropriate production of hormones
Invasion and spread
is achieved by a process called epithelial- mesenchymal transition (EMT) this is a metaplastic process
– a change from one differentiated to state to another state
Malignant facts
Poorly circumscribed
– Strands of neoplastic tissue extend into the normal tissue
– “crab-like” hence cancer
- Often have central necrosis
– Surfaces are often ulcerated - Histologically less resemblance to
parent cell. Not-encapsulated. Invasive. high growth rate
metastasise (spread) often lethal. Produce enzymes (matrix metalloproteases) that degrades extracellular matrix = invasion
Benign neoplasia are
tumours
Malignant neoplasia’s are
Cancers
Types of malignant neoplasias
Carcinoma
– arising from the epithelial tissue
- e.g. gastrointestinal tract, respiratory tract, skin & organs with epithelial-
lined ducts (breast, pancreas, salivary gland, liver). - Sarcoma
– arising from the connective/supportive tissues - e.g. cartilage, bone, muscle, blood vessels, lymph vessels
Protective epithelium tumours
Benign - Papilloma
Malignant - Sqaumous cell carcinoma
Basement epithelium
Malignant - Basal cell carcinoma
Secreting epithelium
Benign - Adenoma
Malignant - Adenocarcinoma
Fibrous
Benign - fibroma
Malignant - fibrosarcoma
Nerve sheath
Benign - neurofibroma
Malignant - neurofibrosarcoma
Adipose
Benign - lipoma
Malignant - Liposarcoma
Smooth muscle
Benign - leiomyoma
Malignant - leiomyosarcoma
Striated muscle
Benign - Rhabdomyoma
Malignant - Rhabdomyosarcoma
Cartilage
Benign - Chondroma
Malignant - Chondrosarcoma
Bone
Benign - Osteoma
Malignant - osteosarcoma
Haematopoietic & lymphoid tissues
Benign - myeloproliferative or myelodysplastic disorders
Malignant - Leukaemia, malignant lymphoma, Hodgkin’s disease (lymphoma), multiple myeloma
Haematopoietic cells
Note the normal cells of the hematopoietic system are able to enter the blood stream and travel around the body.
When these cells have undergone a malignant neoplastic change to become “blood cancer” they are sometimes called “liquid tumours”
Malignant neoplasms of lymphoid cells can be found at
Lymph nodes, spleen, GI tract
Germ cell neoplasia
Neoplastic transformation of toti-potent germ cells
– Haploid: 23, 1N
- Result in neoplasms derived from all three germ layers and can consist of
more than one cell type - Occur in the germ cells of adults
- Germ cells in the testes (spermatozoa)
– Seminoma and non-seminoma “testicular cancer”
– >55y vs ~ 25y - Germ cells within the ovary (oocytes)
– In adult women germ cell tumours are often benign
– dermoid cysts or mature cystic teratomas
Embryonic germ cell neoplasia
Germ cells in the embryo develop at sites remote to
the gonads and these tumours (teratoma) can occur
in embryos/foetus at sites other than the gonads
Teratoma = “monstrous tumour”
Blastoma
Tumours that occur almost exclusively in children less than 5 years old
– Derived from neoplastic transformation of either oligo- or uni-potential stem cells in the embryonic tissue
* These neoplasms resemble primitive embryonic tissues
- The term blastoma is derived from “blast cells” these are the
precursors of the mature cell in that embryonic tissue
– Retinoblastoma
– Nephroblastoma
* Wilms’ tumour
– Hepatoblastoma
– Neuroblastoma
Common adult cancers
Haematological cancers: Lymphoma > leukemia > multiple myeloma
Less common neoplastic malignancies:
Sarcomas, germ cell tumours and childhood cancers (blastomas)
Invasion and metastasis
This process is responsible for the lethal consequences of a malignant neoplasm
- Easy to recognise in epithelial tissues
– Erosion of the basement membrane of the tissue
– Microscopic invasion of cancer cells commonly occurs beyond the gross boundaries of a primary tumor - Difficult to recognise in connective tissue tumours
Metastasis
* Metastases can be the first clinical sign of malignant disease
- Palpable lymph nodes, Bone pain
- Involves three steps or processes:
- Neoplastic cell invasion of surrounding tissue
- Embolization
– enter and travel through blood and lymphatic vessels - Extravasation
– exit the vessel and invasion of new tissue site - Barriers for spread of epithelial neoplasia (carcinoma) are the basement
membrane and the stromal extracellular matrix
How do malignant neoplastic cells spread?
Blood vessels
Lymphatic vessels
Trans-coelomic spread
Lymphatic metastasis
Lymphatic vessels are easier to invade than blood vessels but the neoplastic cells then need to pass through the draining lymph node, which is an additional barrier to distal spread
- Tumour cells settle and grow in the lymph node
- The lymph node will feel firmer and larger than normal
– Poor drainage
– Oedema of surrounding tissue
Trans-coelomic metastasis
Seeding of body cavities by effusion of fluid (ascites) into the peritoneal, pleural and pericardial cavities
- Ascites fluid contains fibrin and the neoplastic cells
- Neoplastic cells grow as tumour “nodules” on the mesothelial surface of the cavity
Prognosis of cancer
Malignant neoplasms (“cancers”) have variable
prognosis (outcome)
– Highly invasive & rapid growth rate
- Treatment options include:
– Surgery, radiotherapy, and drugs (chemotherapy, hormone
therapy, immunotherapy) - Information on tumour type, grade & stage can aid
treatment options - In general, the higher the stage or the higher the grade,
the worse the prognosis
Grading
well-differentiated
– looks very similar to normal tissue
moderately differentiated
– looks something like normal
poorly differentiated
– hardly looks like normal tissue
anaplastic
– virtually no similarity to normal tissue
Staging
Local invasion
* spread within the organ of origin
Local metastases
* Spread to the lymph nodes closest to the organ of origin
Distant metastases
* spread to other organs or to distant lymph nodes
TNM classification (for staging)
T: based on the size and/or extent of invasion through the organ site. (tumour)
- N: indicates the extent of lymph node involvement. (node)
- M: indicates whether distant metastases are present. (i.e. secondary tumours) (metastases)
Staging of malignant neoplasms
Tis In situ, non-invasive (confined to epithelium)
T1 Small, minimally invasive within primary organ site
T2 Larger, more invasive within the primary organ site
T3 Larger and/or invasive beyond margins of primary organ site
T4 Very large and/or very invasive, spread to adjacent organs
N0 No lymph node involvement
N1 Regional lymph node involvement
N2 Extensive regional lymph node involvement
N3 More distant lymph node involvement
M0 No distant metastases
M1 Distant metastases present
Types of DNA mutations that causes cancer
Mutagens
Heritability
Loss of DNA repair
Errors in mitosis
Epigenetics
Environmental Mutagens cause cancer:
Genotoxic
* X/γγRays -> double stranded chromosome breaks
* UV radiation and alkylators in smoke, aflatoxins -> base changes /point mutations
* ROS -> DNA oxidation, variety of mutations
* Oncogenic viruses -> add oncogenic genes/proteins and cause insertional mutagenesis
(activation/ inactivation of genes at the site of viral genome insertion into the host cell DNA)
Inheritance of cancer predisposition gene variants:
Inheritance of specific mutations in specific genes (Pathogenic Gene Variants) increase the
risk of an individual developing specific cancer types during their lifetime
* 5-10% of all cancers, dependent on the cancer type
* Examples:
* RB1, Retinoblastoma syndrome -> Ocular, melanoma, sarcoma
* TP53, Li Fraumeni syndrome -> lymphoma, sarcoma, glioma, breast
DNA repair is essential to reduce formation of cancers during life:
DNA repair -> protective, normal process to repair damage to DNA
* Strand breaks (single or double) and point mutations can be repaired efficiently if detected in
damaged cells before mitosis
Evidence DNA repair is important in protecting from development of cancer:
- 25% heritable mutations in DNA repair machinery genes e.g. BRCA1, TP53
- Mismatch Repair (MMR) defects lead to development of colon, stomach, uterine
cancers (syndromes). - These DNA repair machinery genes are also dysfunctional in sporadic cancers
Mutations can be caused through errors in mitosis:
- Highly mitotic cells are more likely to develop cancers e.g. intestinal epithelium
- Pathologies that lead to high rates of mitosis via repair increase risk of cancer e.g. chronic inflammatory diseases (IBD, NASH)
- Can lead to aneuploidy, having more or less than two copies of (some) chromosomes
- Copy number changes (loss/deletion) are seen in genes that normally pause the process of cell cycle and mitosis e.g. RB1 and TP53
Epigenetics is also important:
*Epi - ‘above’
* Some cancers have very low rates of mutations BUT they’re still cancers
* Can be driven through changes to gene expression through regulation by promoter or histone modifications - methylation and acetylation of DNA bases or histones
* Can be inherited from a cell to it’s daughters - permanent effect
What is a clone?
- A population of daughter cells descended from a single progenitor cell
- Genetically related - but new mutations can be gained, or lost, from a cell leading to tumour lesions being genetically and phenotypically heterogenous
- Common mutations, those present in all tumour cells in a lesion are called ‘clonal’
- Mutations found in a subset of cells in a tumour are ’subclonal’
- Most cancers are descended from a single precursor cell hence they’re
monoclonal
Evidence of clonality
X-inactivation
Antigen-receptor gene recombination
Mitochondrial inheritance
X Chromosome Inactivation
- Females inherit two X chromosomes, two alleles for each gene on the X chromosome, one from each parent
- RANDOM Inactivation of one X in all female cells - ~50% cells in the body will have one inactivated the other ~50% will have the other
BUT - Female tumours have the same X chromosome inactivated
Antigen Receptor Gene (IGH, TCR) Recombination
- During development, in B and T lymphocytes, antigen receptor genes assemble through random splicing of VDJ elements. There
many combinations possible. - If you analyse a blood sample (with mixed lymphocytes in it),
these will have a polyclonal population - containing with different versions of VDJ gene recombination
BUT - In lymphoma, the VDJ gene is monoclonal - indicating that all of the cancer cells arose from a single cell progenitor
Mitochondrial inheritance
- If a mutation is gained in a mitochondrial genome this can become dominant so that all mitochondria in a cell carry the mutation
- Daughter cells derived from that parent cell will all carry the same mutation in the mitochondrial genome
BUT
* In cancers, all cells can carry the same mitochondrial genome. The same effect is seen for early ’driver’ mutations in cancer:
* same driver mutations in a nuclear oncogene in all malignant cells in a tumour
* oncogenic viruses (HPV, HBV) randomly incorporate their genome into nuclear DNA - all daughter cells have the viral genome inserted at the same genomic location
Tumour evolution
- If you take multiple small biopsies of the SAME tumour mass and analyse the genetic and epigenetic information you will detect spatial intratumour heterogeneity - not every tumour cell in a lesion is exactly the same, genetically and phenotypically.
- Cancer cells evolve through collection of gene variants and changes to their gene expression in order to adapt to and survive their environment (stress/treatment).
- Evolution also allows cells to successfully metastasise and seed new sites in the body.
Phylogenetic Tree
Trunk - shared, originating mutations present in every cancer cell - often called
Founder, Clonal or driver mutations in oncogenes or tumour suppressors (key
‘cancer genes’)
Branches - early subclonal but common
mutations
Twigs/leaves - recent, ‘private’ mutations present in a small subpopulation of cells
Forms of Evolution
Selective - In response to external environmental pressures or therapy induced selection. Allows survival of cells that have a proliferative/survival or resistance advantage.
Neutral - Gene Variants, often called passenger mutations, that are non-beneficial to the cell but maintained in genome. This is called Random Drift.
Outcome of Evolution
Clonal tumour cells in a lesion/patient change in proportions in response to selective pressures.
A tumour mass at one time is NOT the same as at another time (Temporal Heterogeneity) -> tumours change and evolve to survive.
How can we follow tumour evolution in patients?
Tissue biopsies throughout clinical care - patient morbidity
* Autopsy analysis - end of life analysis - fixed time point
* Liquid biopsy - minimally invasive analysis of tumour-shed DNA in biofluids
Biofluids biopsy:
Blood, urine, cerebral spinal fluid, abdominal ascites, lung effusions …
Analysis of cells in a biofluid = liquid aspirate/cytology
Cell-Free Tumour DNA (ctDNA) liquid biopsy:
- Gene variants are specific to tumour cells (cancer is a genetic disease!)
- Captures information from all lesions in a patient (including small tumours that might not be visible by imaging or accessible for core biopsy)
- Can track a single ‘driver’ mutation present in every tumour cell in the body to monitor overall tumour mass
- Can identify mutations that can be used to select molecularly targeted systemic treatment
- Can track emergence of resistance mutations in response to treatments
TP53 mutation + EGFR therapy ‘sensitive’
mutation
= driver present in all cells - tracking total tumour burden
EGFR therapy 1
leads to reduction in tumour size but emergence of resistant clone, through a new mutation, that overgrows the
tumour (ctDNA allows early detection)
EGFR therapy 2
again tumour shrinks but
another new resistance mutation occurs and becomes dominant - present in all resistant cells which have a survival advantage
Genetic Changes
- Amplification/Deletion (large scale aneuploidy - chromosomal)
- Single Gene Mutation (point /small insertion-deletion ‘indel’)
- Chromosomal translocation
- Epigenetic methylation
All lead to aberrant activation (switching on) or inactivation (switching off) of specific genes so they function incorrectly (at the wrong time)
Amplification/Deletion
Amplification = extra copies of INTACT genes. Can be caused by:
* increased number of chromosomes (>2) - from errors in cell division/mitosis
* Formation of “double minute” chromosomes - extra fragments of DNA carrying specific (onco)genes outside of the nuclear genome. Can be reincorporated into cell
chromosomes.
Leads to production of more protein by having extra copies of genes. Deletions can also occur during mitosis/aberrant DNA repair leading to loss of specific (tumour suppressor) genes = less protein
Partial Gene Deletions
e.g. Oncogene - Epidermal Growth Factor Receptor (EGFR)
* With no ligand, EGFR inhibits itself
* Dimer binds ligands (EGF and TGFa)
* Activates downstream signaling through
phosphorylation via the intracellular tyrosine kinase domain -> cell proliferation
In cancer, EGFR signalling is activated
- gene amplification -> more protein
- deletion of exons 2-7 -> removes inhibitory ligand binding domain -> ON tyrosine kinase activity without ligand binding (activating mutation)
Translocations
Rearrangements of genetic material to juxtapose gene promotors/regulatory elements to drive inappropriate gene expression (activating mutations).
Example:
* Translocation of CCND1 gene downstream of an active IGH enhancer element
* Switches ON CCND1 gene -> excess cyclin D1 protein, deregulation of cell cycle at R point
* Occurs in Mantle Cell Lymphoma
Evidence of the effects of excess oncogenes:
Transfection of immortalized mouse fibroblasts with ‘cancer mutations’ leading to fully transformed cells:
* are growth factor independent
* have 3D growth (disorganised)
* lose Anoikis (an apoptotic death triggered by detachment from the ECM/basement membrane) -> allows anchorage independent growth
* form tumours in vivo (invasive/metastatic)
Cause of retinoblastoma
Retinoblastoma often caused by inheritance of one
mutation in RB1 gene and acquisition of a second mutation which switches off its function (regulating cell cycle and proliferation)
Alfred Knudson’s TWO-HIT hypothesis for inactivation of
tumour suppressor genes
- First hit - inheritance or somatic mutation of one allele
- Second hit - somatic mutation/deletion or epigenetic inactivation of second allele
Causes total loss of RB1 protein (pRb). pRb function is lost in 25% of all cancers through inactivating mutation or epigenetic changes (promoter methylation).
Oncogenes
Overactive proteins
EGFR, RAS, BRAF, CCND1
Tumour Supressor Genes
Inactivated proteins
RB1, TP53, BRCA1/2, APC
Growth factor cell signalling proteins
EGFR, RAS, BRAF
Cell cycle proteins
CCND1, RB1
DNA repair proteins
TP53, BRCA1/2,
Cell adhesion molecule
APC
Cell cycle G0
G0 - exit the cell cycle - Quiescence OR - Re-enter the cell cycle and prepare for
DNA duplication
Cell cycle G1
G1 - Gap 1 - cells detect growth factors, space,
substrate attachment (environment) and either:
- G0
Cell cycle - R
- restriction point in the G1 phase - checkpoint to continue cycling - often ignored in cancers due to incorrect activity of the checkpoint proteins
Cell cycle - S
DNA synthesis - DNA is replicated
Cell Cycle - G2
Gap 2 - more checkpoints for completion of
DNA replication. Preparation for mitosis.
Cell cycle - M
ends with cytokinesis, production of two daughter cells
Activator and inhibitor of the cell cycle
-Cyclins and Cyclin Dependent Kinases (CDK) are regulated by phosphorylation
-Cyclins bind to and increase the kinase activity of CDK allowing progression of the
cell cycle
Cyclin-CDKs regulate
- the R restriction point
- the G1/S transition
- induction of DNA synthesis in S phase
- the G2/M transition
- The cell cycle is paused by the activity of inhibitors of the CDKs (e.g. p21cip) and checkpoint proteins including pRb and p53
Growth factor signalling
- Promotes entry into the cell cycle
- Relieves blocks on cell cycle progression (promoting replication)
- Prevents apoptosis
- Enhances synthesis of components (nucleic
acids, proteins, lipids, carbohydrates) required for cell division
EGF
EGF, epidermal growth factor - Mitogenic, stimulates epithelial cell migration, stimulates formation of granulation tissue.
Oncogenic growth factor signalling
- Increased levels of growth factors (sometimes
different ones in metastases) - Permanent activation of growth factor receptor
- Increased levels of growth factor receptor
- Abnormal signal transduction (e.g. Active Ras)
Overall effect - advances cells through the cell cycle
Growth factor signalling
Growth factor binding to the receptor
e.g. EGF or TGFa to EGFR induces
* receptor dimerisation,
* tyrosine kinase activation
* phosphorylation of the receptor
Oncogenic mutations lead to
constitutive activation of the tyrosine kinase receptor signalling, in absence of the growth factor.
Ras Pathway
- Ras is an intracellular protein that is central to integrating GFR signals
- GFR activation -> Ras binds GTP and recruits and activates signalling proteins (e.g. Raf, PI3K)
- Signalling stops when Ras converts GTP to GDP+phosphate (via its GTPase activity)
- Cancers with mutant Ras have reduced GTPase activity -> remains active.
Ras activates transcription of CCND1, cyclin D which activates CDK4 to stimulate cell cycle progression.
Excess oncogenic cyclin D or CDK4 (by activating mutations/amplification) occur in cancers.
Retinoblastoma Protein (pRB)
The RB protein exists in two forms during the cell cycle
* Weakly phosphorylated - at G0 and G1 - active - halts cell cycle at the Restriction point by suppressing transcription factor signalling
- Strongly phosphorylated (ppRb) - during rest of cell cycle - inactivates the protein so it does not signal and therefore cell cycle progresses
Inactivating phosphorylation of pRB is by CyclinD-CDK4
Loss of RB protein (e.g. by gene deletion/inactivating mutation/methylation) leads to uncontrolled cell cycle progression (skipping the G0/G1 R checkpoint)
Oncogenes (activation of proteins)
KRAS - Ras GF signalling activated
PIK3CA - PI3K GF signalling activated
Tumour suppressor genes - (deactivation of proteins)
TP53 -> p53 -cell cycle checkpoint lost
CDKN2A -> p16 - inhibitor of CDK4 lost
1mm to 1cm cells (1 mil to 1 bil)
<5 doublings
Apoptosis
Most tumours have high rates of proliferation AND apoptosis (cell turnover)
* Caused by an inhospitable environment - lack of oxygen, nutrients
* Immune attack!
* Cell signalling can be dysregulated driving apoptosis
Although often non-lytic necrotic cell death occurs, large-scale ‘tidy’ apoptosis
can trigger wound healing
Apoptosis occurs at any point in the tumour development to promote the initial tumour growth and regrowth after cytotoxic therapy
Apoptosis drives wound healing by
Effector caspase-3 cleaves phospholipase A2 (iPLA2) from cell membrane lipids.
Produces arachidonic acid which is metabolised by COX2 into prostaglandin (PGE2) -> ++ cancer cell proliferation
Exposed phosphatidylserine (PS) on apoptotic bodies (‘eat me’), ingested by macrophages which then release
* VEGF -> angiogenesis
* MMPs, PDGF, TGFß -> extracellular matrix remodelling/fibrosis
Suppression of apoptosis
Suppression of apoptosis is important at all stages of cancer from initiation to progression to treatment resistance.
Early suppression of apoptosis:
- At the premalignant stage
- Extends lifetime of cells so allows accumulation of potential ‘driver’ mutations
- Alters normal tissue homeostasis resulting in over production of cells (too
many - hyperplasia)
Suppression of apoptosis:
- Supports invasion (malignancy)
- Allows suppression of anoikis - cells can survive without attachment to
basement membrane - Cells can therefore move - invade tissues (migration) - supporting metastasis
- Supports metastasis
- Allows cells to survive in the inhospitable environment of the blood circulation (e.g.
when receiving pro-apoptotic signals from immune cells (e.g. FasL) - Allows cells to be resistance to therapies such as hormonal treatments that are used to kill cancer cells
Death Receptors
- Extrinsic apoptosis is driven by external signals detected
by cell surface receptors e.g. Fas Receptor - Fas receptor binds to extracellular FasL (ligand)
- Fas receptor binds an intracellular adaptor molecule FADD via a ‘death domain’ then activates an initiator caspase
- The initiator caspase activates an effector caspase which leaves target proteins triggering the cell apoptosis process.
Switching off death receptor pathways in Cancer:
Fas receptor and FADD can be switched off by mutations in the death domain (inactivated protein) or through promoter methylation (less transcription)
- Initiator caspase expression can also be
switched off in cancers by methylation
Leads to:
* resistance to pro-apoptotic signals
* death of tumour infiltrating
lymphocytes cells by secretion of FasL from resistant cancer cells (counter- attack).
BCL-2
Key sensor of cell stress.
It is an ANTI-apoptotic protein that is overexpressed by chromosome translocation to the IGH locus in B cell follicular lymphoma. This
oncogene extends the life of B cells but does not affect the cell cycle (proliferation rate).
BCL-2 and Bax
Regulate the movement of pro-apoptotic proteins like Cytochrome C from mitochondria so they do not interact with and activate the APAF-1 apoptosome.
- BCL-2 closes membrane pores (no release of CytC, anti-apoptotic),
- Bax opens membrane pores (release of CytC, pro apoptotic)
- Oncogenic, Anti-apoptotic BCL-2 is overexpressed in 50% of all cancers
- Activating mutant EGFR protein (delEx2-7) can also induce Anti-apoptotic BCL-XL expression
(as well as activating the cell cycle) leading to treatment resistance - PRO-apoptotic Bax, a tumour suppressor gene, is inactivated in colon and stomach cancers
- LOTS of ways a cancer cell can avoid apoptosis!
p53
- p53 protein is encoded by TP53 gene
- It is the most inactivated protein in cancer, acts as a
tumour suppressor gene (point mutations or gene
deletions) - It is mutated in families with inherited Li Fraumeni
cancer Syndrome - It normally regulates apoptosis (pro-apoptotic) and
is a cell cycle checkpoint protein upon detection of
DNA damage (an adaptive stress response to
genotoxic damage)
Activation of p53
- p53 is activated by DNA damage (by carcinogens
or chemotherapy) or abnormal cell cycle (Ras
mutation, pRB inactivation) - Activation of p53 leads to regulation of the cell cycle and/or activation of apoptosis via transcriptional programmes
- p53 will pause the cell cycle to allow DNA repair,
if too damaged it will initiate apoptosis.
p53 regulation
- p53 is normally kept in check by MDM2 which
binds and targets it for destruction - p53 regulates itself by triggering the expression of
MDM2 in a feed-back loop, keeping it’s activities under control once the emergency stress is resolved
Normally activated p53
induces transcription of
proteins that
- arrests the cell cycle at G1 by inducing expression of p21cip1 an inhibitor of CyclinE-CDK2 allowing DNA repair
- Failure of repair or excessive DNA damage
promotes apoptosis via PRO-apoptotic Fas and Bax
p53 in cancer
Mutant/deleted/inactivated p53: p53 dependent genes no
longer transcriptionally activated
Gene amplified MDM2: too much MDM2 protein targets
p53 for destruction
->
* No cell cycle arrest
* No DNA Repair
If cells survive:
* accumulation of mutations (DNA damage not repaired),
* proliferation of cells (loss of cell cycle checkpoint),
* malignant transformation,
* expansion of the tumour mass,
* chemotherapy can select for cells with p53 mutations
that are resistant to apoptosis
To migrate, cancer cells must:
- Detach from one another,
- Secrete enzymes that breakdown underlying basement membrane and
other Extracellular Matrix (ECM), - Become motile (change shape)
- Survive on different ECM surfaces with different growth factor signals.
Cell-Cell adhesion
- Epithelial cells are organised and orientated (apical, basal), aligned along a basement membrane
- Cells adhere to one another via adherens junctions (AJ) consisting of cadherin and
catenin proteins - In ‘epithelial’ cells, the adhesions are strong and use E-Cadherin
EMT:
epithelial to mesenchymal transition is an essential process that allows epithelial-like cancer cells to gain a migratory and invasive mesenchymal phenotype.
It occurs by trans-differentiation of epithelial cells into mesenchymal cells in response to cellular signals. There are ‘intermediate’ cells.
EMT is triggered by:
IRREVERSIBLE (on -> off)
- Somatic or inherited DNA mutations in E-
cadherin/α-catenin genes - Seen in hereditary diffuse gastric and breast
cancers REVERSIBLE (like a dimmer switch) - Response to external growth factors (GF) or cytokines from inflammatory cells - often
surrounding the tumour causing a ‘leading edge’ of
migratory cells. - These transiently induce transcription of repressors
of E-cadherin (e.g. Snail protein)
EMT in mesenchymal cells
- Adhesions have N-cadherin instead of E-cadherin which is a weaker interaction between cells
- Cells express Snail protein which represses E-cadherin
- Cells express urokinase plasminogen activator (uPA)
receptor (uPAR) on the surface which cleaves plasminogen into active plasmin - Express Membrane Type 1-MMP on the surface that
cleaves extracellular matrix - Plasmin and MT1-MMP activates MMPs that cleave ECM and releases growth factors, supporting cell survival
Mesenchymal state cancer cells
- Detach from one another
-Loss of adherens junctions through downregulation
of E-cadherin via Snail (a transcriptional repressor) - Secrete enzymes that breakdown basement
membrane and ECM.
-MT1-MMP expression, uPA (from fibroblasts)/uPAR
cleaves plasminogen into plasmin -> MMP activation - Become motile
-Remodelling of intracellular actin changes cell shape - Survive on different ECM surfaces with different
growth factor signals
-Growth factors and ECM chemotactic peptides
released by MMPs
EPITHELIAL cells have/are
Apical-basolateral
Strong AJ cell-cell adhesion
- E-Cadherin
EpCAM, Cytokeratins
Immobile
MESENCHYMAL
Front-back polarity
Weak AJ cell-cell adhesion
- N-Cadherin
Fibronectin, Vimentin, Snail, Proteases
Motile - invasive, stem-like, anoikis and therapy resistant
Stages of EMT
*E and M are not two binary states within a cell (ON/OFF, E or M)
* E and M cells can occur within the same tumour lesion (often M at the invasive edge of a tumour)
* There are intermediate stages with a mixture of phenotypes (EMT, MET)
* Snail protein is key to switch states by altering epigenetic (methylation and acetylation)
‘marks’ on specific gene promoters (binding to DNA and histones)
In EMT: Epithelial genes (E-cadherin) switched OFF, Mesenchymal genes (Fibronectin, Vimentin)
switched ON by Snail. Remove Snail, reverse the process.
Examples of factors that modulate the process of EMT and cancer progression
- Adipocytes and Macrophages release IL-6 which allows detachment and loss of cell polarity
- Tumour-associated Fibroblasts release HGF (hepatocyte growth factor) which induces
invasion into blood/lymphatics - Activated platelets in blood secrete TGFß which allows cell survival in the circulation and
stemness to support colonisation
Angiogenesis
Clinically, in ductal breast cancers, angiogenesis supports progression:
* Progression from hyperplasia -> dysplasia -> angiogenic carcinoma in situ -> invasive ductal carcinoma
* Angiogenesis occurs early as an in situ lesion, allowing invasion out of the duct
* Highly vascularized tumours are associated with poor prognosis as they allow ‘early’
metastasis/movement of cells
Tumour progression
Experimentally, angiogenesis supports progression:
* Viral protein (TAg) immortalized mouse pancreatic islet cells (represses expression of p53 and pRB) co- cultured in vitro with endothelial cells.
- (Sometimes) tumours develop through stages Immortilised -> Hyperplasia -> Angiogenic Hyperplasia
-> Carcinoma in vivo.
Angiogenesis supporting progression experiment
+IGF2 causes hyperplasia of 50% of cells (proliferation)
* +MMP-9 in 10% cases - induces endothelial cell ingrowth (angiogenesis)
* Loss of E-cadherin allows cell invasion of 2% cells (invasion)
Interpretation of experiment findings:
- Cancer cells communicate with ECs by secreting diffusible pro-angiogenic
factors (MMP-9 in this case) - The angiogenic factors need to be induced at a specific time during tumour development to promote the
angiogenic islet phenotype - The angiogenic switch is early and is a prerequisite for invasive (malignant) growth.
Angiogenic triggers
Metabolic stress - Hypoxia, low pH, low glucose
Inflammation - cytokines
Acquired cancer cell mutations - Gene regulation, p53 -, RAS +
Hypoxia
- New blood vessels are essential for cancer cell
survival - Cells must be <200μm from a vessel to get oxygen
from the blood - Tumours have disorganized, leaky vasculature and
can outgrow the blood supply causing areas of hypoxia (oxygen-deficiency) - Hypoxia induces stress responses (HIF1 and p53
signals) to induce new blood vessel formation
Hypoxia cancer reaction
- Induces an aggressive, invasive phenotype
- Selects for an aggressive genotype e.g. p53
inactivating mutations provide anti-apoptotic survival
signal - Reduces efficacy of radiotherapy (oxygen is required for damage)
- Reduces efficacy of most chemotherapy (hypoxic cells
exit the cell cycle - quiescence - causing resistance,
and lack of blood supply leads to poor drug delivery)
Sprouting angiogenesis
Process:
* Stimulated endothelial cells degrade basement membrane, change shape
(motile), and invade tissue stroma
* Form a column of cells with a
migratory tip at the leading edge followed by a stalk of proliferating and differentiating cells which assemble into a tube with a lumen
* Tubes coalesce into capillary loops
Co-Option
Process:
* Use of existing blood vessels by growing around them for a short time.
* The vasculature may eventually collapse
which causes a hypoxic tumour. Tumours are characterized by large scale cell death.
* Surviving cells may induce other forms of angiogenesis due to hypoxia.
* e.g. Glioma in the brain.
Intussusception
Process:
* Columns of tumour cells grow into a pre-existing vessel causing them to remodel and expand due to physical
force.
* Essentially splitting of pre-existing blood vessels.
Vascular Mimicry
Process:
* Trans-differentiation of cancer cells allows them to express characteristics
of endothelial cells (stem-like nature)
* Cancer capillaries can be lines with a mixture of cancer cells and stromal endothelial cells.
* e.g. Colon cancer - 15% of vessels are mosaic allowing shedding of cancer cells into the circulation (Million cells/gram tumour/day!).
Vasculogenesis
Process:
* Formation of new blood vessels de novo (from new) in the absence of pre-
existing vessels.
* Requires the differentiation of stem cells into endothelial cells, pericytes and vascular smooth muscle cells.
* Tumour cells (via EMT)
* Bone Marrow precursors
(angioblasts)
* Differentiation is regulated by the levels of PDGF (Muscle) and VEGF (EC)
Anti-Angiogenics
- VEGF binds to growth factor receptors (VEGFR) on
endothelial cells and activates receptor tyrosine kinase
pathways -> cell cycle, proliferation - VEGF is produced by cancer cells and inflammatory
cells - Overexpression leads to abundant, immature, leaky
vessels -> Inflammation + Oedema - Inhibition of angiogenesis/vasculogenesis -> reduces routes of invasion, nutrients etc.
- Cells can be resistant if they have mutations in downstream proteins in the signalling pathway e.g. Ras
- Cells can also form vessels via another, non-VEGF pathway.
Metastasis
- The process by which tumours spread from their sites of origin (primary) to distant sites (secondary, metastases)
- Presence of metastasis is a poor prognostic factor for patients (high
stage disease) - Metastasis is responsible for 90% of mortality - due to causing blockages, use of nutrients, increasing risk of infections, causing
cachexia etc.
Different cancer types have
Have different metastatic potential
Metastasise to different secondary sites
- organ(o)tropism due to chemotactic signals or adhesion to specific cell types via integrins
*simple anatomy - ‘drainage’
Seed and Soil Theory of Organotropism:
Theory suggests that the cancer cell needs to localise itself to a site which can support its growth
- pre- metastatic niches in organs that are ‘similar’ cells of origin or have been ‘prepared’ through secreted signals modifying the microenvironment.
What routes do cancer
cells use?
- blood circulation
(hematogenous) - lymphatics
- across hollow organs or
along tissue boundaries
(transcoelomic)
Metastatic Spread
- Local invasion (via EMT)
- Intravasation (blood and lymph)
- Transport in the blood (hematogenous spread)
- Extravasation
- Colonisation
Intravasation
Perivascular TAMs secrete EGF to attract motile tumour cells (via binding EGFR) towards vessels
Cancer cells can move singly or in clusters
Endothelial cells lining capillaries are induced
by
* TAM derived TNF
* Cancer cell derived TGFß and MMP1.
Induction causes vascular-endothelial (VE) adherens junction disassociation and EC retract allowing cancer cells to transmigrate in between/around cells (Paracellular).
Other signals induce contraction of
actomyosin actin-myosin cytoskeleton filaments in EC
Creates pores IN the endothelial cells to
allow transcellular migration of cancer cells into the blood vessel.
Transport in the blood (hematogenous spread)
- Cancer cells in the blood are called circulating tumour cells (CTC)
- These CTCs survive in the circulation through formation of platelet emboli
- Platelets link between macrophages and tumour cells, a structure that protects from shear force (fluid flow)
- Induces further EMT by platelet secretion of TGFß and
cytokines - Platelets lay down fibrin in presence of tumour cell secreted protease thrombin (cleaved from fibrinogen) to protect cancer cells from surveilling natural killer cells.
- Rapid tumour growth causes secretion of cell clusters of invasive mesenchymal CTCs
(Fibronectin and N-Cadherin+) - Regression of tumour growth, after successful therapy, is associated by presence of single, epithelial CTCs in the blood (EpCAM, cytokeratin, E-cadherin+)
- Recurrence of a tumour, shows an increase in number again in mesenchymal- like CTC clusters
Extravasation
- Cancer cell releases IL-8 to activate neutrophils which bridge between EC and cancer cell/platelet emboli
- Expression of ICAM-1 (on EC and Cancer cell) and P-selectin (on EC and platelets) allows adhesion via neutrophil LFA1 integrin and sialyl-Lewis-X proteins
- Cancer cell releases
chemokines to recruit and
activate monocytes that
release VEGF - VEGF dismantles adherens
junctions, EC retraction
allowing transmigration into
organ tissues
Colonisation
During the process of colonisation, circulating cancer cells (CTC) die en mass
* Passing through endothelial cells (cell injury)
* lack of survival signals (growth factors)
* lack of supportive structure (anoikis)
* tumour cell killing by neutrophils (immunity)
Surviving cells pass into tissues as Disseminated Tumour Cells (DTC). <1% DTC form metastatic tumours. The process of metastasis selects for cells that can survive all steps.
Tumour cells and dormancy
- Tumour cells that spontaneously disseminate from a mass had a lower survival rate than those injected directly into the blood (intravasation kills cells) BUT they extravasate more quickly and can survive for longer at sites of colonization through stem-like dormancy.
- Dormancy is activated by stromal cells (macrophages) - the stroma in sites of metastasis must produce factors that enable the cancer cell to survive and respond to signals from the cancer cells correctly.
- Non-malignant stromal cells are co-opted by the tumour to support survival, enhancing stemness and immune evasion.
- DTCs can be dormant, cells that are challenging to treat with cytotoxic therapy,
leading to treatment failure.
- Cellular/Single cell dormancy
- Exit from the cell cycle (quiescence)
- Caused by lack of GF, adhesion, stromal signals
e.g. In lung cancer, Bone Morphogenic Protein release by stromal cells can suppress proliferation of stem-like cancer cells
e.g. Dormant stem-like DTC in bone marrow adjacent to blood vessels are suppressed by Thrombospondin (TSP) released by vascular endothelial cells.
- Micrometastatic cell dormancy
- Clusters of dormant cells
“tumour mass dormancy” - Balance of proliferation and death
a) Angiogenic: Lack of angiogenesis, results in a balance between proliferation and cell death
b) Immune: Killing of susceptible cancer cells by immune cells (Th1 derived IFNg and IL-12). Supports selection of cells that evade immune surveillance.
c) Therapy induced: e.g. Hormone deprivation therapy in breast, prostate cancers
shrinks tumour mass to a point where proliferation and apoptosis are balanced.
Dormant cells might not be clinically detectable = “minimal residual disease”
Reactivation of dormant cells
Due to a change in microenvironment that supports cell proliferation
Angiogenesis
- Endothelial sprouting and fibroblast secretion of ECM proteins periostin and tenascin C in breast cancer
- Recruitment of bone marrow progenitor stem cells for vasculogenesis
Immune
- Downregulation of tumour cell antigen presenting MHC-I (immune evasion -> cell survival)
- Recruitment of immune suppressive cells Myeloid Derived Suppressor cells or T-regs
Achondroplasia
an autosomal dominant mutation in the fibroblast growth factor receptor
gene 3 (FGFR3) causes abnormal Impaired growth of cartilage
X/γγRays
double stranded chromosome breaks
UV radiation and alkylators in smoke, aflatoxins
base changes /point mutations
ROS
DNA oxidation, variety of mutations
Oncogenic viruses
add oncogenic genes/proteins and cause insertional mutagenesis
