306BMS Cancer Biology Revision PDF

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306BMS Cancer Biology RevisionSection BPat’s MaterialLecture 1Cancer BiologyNeoplasm: mass of cells proliferation at an inappropriate rate (neoplasia  new growth, lump, tumour)What is cancer?Common disease that will affect 1 in 3 people over their lifetime worldwide Group of diseases that result in...

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306BMS Cancer Biology Revision Section B Pat’s Material

Lecture 1 Cancer Biology Neoplasm: mass of cells proliferation at an inappropriate rate (neoplasia  new growth, lump, tumour)

What is cancer? Common disease that will affect 1 in 3 people over their lifetime worldwide Group of diseases that result in spread of mutated cells throughout the body (Pecorino 2012)

Clinical diagnosis Benign  Non cancerous tumour  Cytologically normal  Contained by capsule of connective tissue  Localised: no spread to distant sites  Damage: local pressure or obstruction  Surgical removal Malignant  Cancer  Cellular abnormalities  increase in cell number, variation in shape and size, increase in nuclear size and staining density  Invades/destroys  Metastasise to other locations  cause secondary tumours  Lethal if untreated

Benign tumours can have serious effects     

Brain tumour: increase intracranial pressure Pituitary glands at base of brain produces many hormones  each produced by different cell type Benign tumours – overproduction of 1 or more hormones May press on/damage normal cells  loss of function Pressure on optic nerve  blindness

Why are malignant tumours life threatening? Physical obstructions Invade other organs Compromise function Compete fiercely with healthy tissue for nutrients & oxygen

Diagnosis Grading:  Degree of differentiation  Microscopic examination  Histological (structure of tissue) & cytological criteria  Histopathologist (H&E stain) Staging:  Extent of tumour spread  Morphological assessment

 T= tumour size  N = lymph nodes involved  Molecular markers Normal tissue: population of cells organised into tightly-knit communities  optimal size, tight association, no invasion Cancer cells break the communal rules of tissues Cancer cells do not adhere to the extracellular matrix  anchorage independence

Portrait of Cancer       

Abnormal growth Inappropriate division Local invasion No contact inhibition Loss attachment Metastasis Histopathology: cellular transformation

Tumour evolution: carcinogenesis Dynamic, multi-step: gradually acquires new characteristics  initiation and progression  Mutation in a single cells  competitive advantage – more rapid proliferation than neighbours, growth independent of external growth factors  Limitation  nutrients + O2  stimulate and sustain angiogenesis (formation of new blood vessels)  Over time  descendants acquire additional mutations: genome instability, overcome regulation to restrain growth, suppress apoptosis, immortal  Invade tissues & establish distant secondary tumours  metastases Microarray analysis: compare gene expression patterns of metastases and primary tumours  search for metastases-associated genes

Metastasis Transfer of disease from one organ or part to another not directly connected to it  Stimulate angiogenesis as malignant tumour needs nutrients  New blood vessels in tumour not well formed  easily damaged  Tumour cells break away from original tissue  Attach to wall of a blood or lymph vessel  Secrete digestive enzymes  Cross the wall at breach  into lumen of penetrated vessel  Creep/tumble inside blood vessel  survive  Arrest  move out of lumen in capillary bed of distant organ  Growth  start new secondary tumours Many tumours eventually release cancer cells that migrate to distant sites in body to form secondary tumours called metastases

mFISH Multicolour fluorescence in situ hybridisation (mFISH)  paint each metaphase chromosome with distinct colour

Hallmarks of Cancer    

Growth signal autonomy Evade growth inhibitory signals Avoid immune surveillance: recognises and destroys cancer cells Unlimited replicative potential: cancer cells maintain length of telomeres

 Cells migrate and invade  Induce angiogenesis  Evade cell death  apoptosis Enabling characteristic: Genome instability and mutation  faulty DNA repair pathways

Properties of transformed cells         

Altered morphology  rounded shape, refractile in phase-contract microscope Loss of contact inhibition  ability to grow over one another Ability to grow without attachment to solid substrate  anchorage independence Ability to proliferate indefinitely  immortalisation Reduces requirement for mitogenic growth factors High saturation density  ability to accumulate large numbers of cells in culture dish Inability to halt proliferation in response to deprivation of growth factors Increased transport of glucose (Warburg effect) Tumorigenicity

Risk 1 in 3 diagnosed with cancer during life time Usually older (55+) 200 types >50% cancers: lung, breast, prostate, colorectal

Geographical variations in rates Age standardised incidence rate of all cancers (except non-melanoma skin cancer) is 2.5 times higher in more developed countries  Highest rates in North America, lowest in Africa and Asia] Disparity suggests certain cancers caused by ‘Western’ lifestyle: earlier onset of smoking, diet, sedentary 

Migrant studies Migration effects show environmental factors are major determinants of specific cancer risks  Compare age standardised incidence rates in populations moving from country of low to high rates  Risk of breast cancer for Japanese women in Japan quarter that of white women in US  Migrant Japanese women in US have similar rates to US whites  Rates in Japan and China rising rapidly Influence of Diet:  Stomach cancer (12.8% cancers in Japanese females)  minor cancer in US  Risk of developing stomach cancer for Japanese women who migrate to US decreases if adopt American diet, not if they retain Japanese diet

Cancer is a multi-step process To transform a normal cell into a tumorigenic (malignant) cell requires multiple mutations How are these tumour-promoting mutations acquired? Inherited  familial cancer syndromes Spontaneous/sporadic  errors in DNA replication, repair or chromosome segregation Carcinogenic agents  Identify environmental (behavioural, reproductive and lifestyle factors) and other carcinogens  Determine specific mutations induced  Elucidate pathways they affect

Smoking as a Carcinogen

Tobacco is single most important risk factor for cancer: Caused 22% of all cancer deaths (1.7million in 2008) 71% of lung cancer deaths (1 million in 2008) Long latency period  rates of lung cancer reflects patterns of smoking decades before  81 carcinogenic compounds in cigarette smoke  Benzo[a]oyrene (BP): lipophilic polycyclic aromatic hydrocarbon  Liver enzyme metabolises BP to other highly mutagenic species  Mutations induced: G to T mutations

Mutations induced by BP MAP DNA adducts (chemically modified bases) of TP53 gene induced by BP diol epoxide Match distribution of p53 mutations in lung tumours from smokers Direct casual link between a defined carcinogen and lung cancer Mutation signature

Genetic Polymorphisms and variable expression   

Liver cytochrome enzymes metabolise chemicals  activate carcinogens Expression of CYP1A1 varies 50 fold in human lung tissue Individual variation in carcinogenic ‘dose’ among smokers

Cancer promotion by non-mutational mechanisms     

Viral infection eg. Human papillomavirus (HPV) and cervical cancer High levels of proteins E6 and E7 from viral genome Bind and inhibit pRB and p53 respectively Inactivating 2 major tumour suppressor proteins No mutation of genes in host cell

Prevention: Cervical cancer vaccine Vaccination against strains of human papilloma virus Cervarix (cheaper; bivalent: HPV 16 & 18 years) vs. Gardisil (quadrivalent; also 6 & 11; also protects against genital warts) Girls aged 12-13 from September 2008

Personalised Treatment: The Lung Mutation Consortium        

14 US hospitals Patients with advanced lung cancer Genomic tumour profiling Use in therapeutic decision -making Report: 700 patients 10 specific ‘driver’ mutations Presence of one of these in 64% cases In 28% of these  specifically targeted therapy for their driver mutation - Oncogenic driver mutation identified with targeted therapy  3.5 years median survival - Oncogenic driver mutation identified without targeted therapy  2.4 years median survival - No driver mutation identified  2.1 years median survival

Molecular diagnosis: distinct diagnostic features and clinical outcomes Cancer seen less as a disease of specific organs, more as one of molecular mechanisms caused by mutation of specific genes

Biopsies: future is liquid  

Blood, lymph, skin Others  invasive procedures

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Blood test i.s.o biopsy Circulating tumour DNA (ctDNA) Liquid biopsies: diagnosis and monitoring treatment Detect before any symptoms Ultra deep sequencing machines: reads DNA in a sample many times  picks up rare signals (ctDNA)

Locating tumours Imaging biomarker distinguishes prostate cancer tumour grade  restriction spectrum imaging (RSI) imaging biomarker that enhances ability of MRI to differentiate aggressive prostate cancer from lowgrade or benign tumours and guide treatment and biopsy Mutations in genes that stimulate tumour progression  Oncogenes: code for proteins that promote cancer (mutation in proto-oncogenes leads to overactivity or overexpression)  Tumour suppressor genes: code for proteins that restrain cell proliferation (negative regulator)

Lecture 2 Oncogenes and EGFR pathway Cancer is the result of somatic cells acquiring genetic changes that confer six general features:  Independence of external growth factors  Insensitivity to external anti-growth signals  Ability to: avoid apoptosis  Replicate indefinetly  Trigger angiogenesis  Invade tissues and establish secondary tumours

Peyton Rous Launched modern cancer research with hens Rous’s protocol for inducing sarcomas in chickens:  Chicken with sarcoma in breast muscle  remove sarcoma and break up into small chunks of tissue  grind up sarcoma with sand  collect filtrate that has passed through fine-pore filter  inject filtrate into young chicken  observe sarcoma in injected chicken

Retroviral oncogenes Rous sarcoma virus Avian leukosis virus Transforms chicken embryo fibroblasts in Replicates in same cells but does not induce culture and induces large sarcomas > transformation inoculation into chickens Genomic RNA: 10kb Genomic RNA: 8.5kb Has genetic information responsible for Lacks this informations transformation of infected cell Deletion and ts mutants which cannot induce transformation Oncogenes src  encodes a protein: first Lacks this gene protein-tyrosine kinase identified The difference between two viruses:  Rous sarcoma virus (RSV) isolated from chicken sarcoma (cancer of any connective tissue)  Closely related avian leukosis virus (AVL) The ability to transform cells resides in this one gene

src

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src related sequences in DNA of uninfected chicken cells Cellular version (c-src or proto-oncogene) Highly conserved gene of all vertebrates Viral form of src is a mutated version of c-src

Other retroviral oncogenes: rasH  induces Harvey sarcoma in the rat rasK  induces Kirsten ssarcoma in the rat raf  induces murine sarcoma Encode proteins that are key components of signalling pathways that stimulate cell proliferation Nonviral oncogenes in chemically transformed cells

Oncogenes in the DNA of cancer cells Chemically transformed mouse fibroblasts  DNA  transfection using calcium phosphate coprecipitation procedure  normal mouse fibroblasts infected  formation of a focus of morphologically transformed cells  injection of cells into mouse host  tumour

Epidermal Growth Factor signalling pathway In normal cells and cancer cells The signal transduction pathway of EGF is characterised by the sequential steps of growth factor binding, receptor dimerization, autophosphorylation, activation of an intracellular kinase cascade, activation of transcription factors and regulation of gene expression. MAPK – mitogen activated protein kinase Growth factor  receptor  intracellular signal transducers  signal from cytoplasm  nucleus  mitogenic effect, stimulate cell growth, trigger cell proliferation Oncogenes codes for mutated protein tyrosine kinase receptor  target for development of specific anti-cancer drugs  Extracellular dimerization domain (EGFR side chain slides up into place)  Phosphorylation results in conformation change  Dimerization and autophosphorylation  intermolecular phosphorylation  Another phosphorylation  Conformational change (sliding up of EGFR exposes binding sites allowing the two to bind causing phosphorylation)  Inhibits ligand binding and kinase activity  De phosphorylation by tyrosine phosphatase  Binding of negative regulators to kinase domain  Receptor endocytosis and degradation Binding of ligand induces dimerization which activates cytosolic kinase domain  Propagates the signal

Ras  Rat Sarcoma Virus Lipid moiety allows Ras to anchor on cell membrane The SH2 domain of GRB2 (100 amino acids) bind to specific short peptide sequences with phosphotyrosine residences on EGFR SOS upstream activator converts Ras to active state through the exchange of GDP to GTP GRB2: adaptor  physical bridge between receptor and SOS Ras activates PI3K and RAF. RAF activates MEK which activates MAPK. MAPK causes the phosphorylation of transcription factors resulting in a mitogenic response by the cell  active growth and division

Active PIP3K activates PIP2 to PIP3 which binds PDK-1 and AKT. AKT is phosphorylated causing downstream effectors: serine/threonine kinases  initiate transcription causing same mitogenic response by cell for active growth and division

Binary switch between active and inactive form of Ras Upstream stimulatory signal and Ras activation triggered by GEF Inactive Ras + GDP  GEF (Guanine nucleotide exchange factor)  Active Ras + GTP  Downstream signalling GTP hydrolysis by GTPase and Ras inactivation induced by GAP Active Ras + GTP  GAP  Pi + Inactive Ras + GDP Blockage of active Ras and GAP caused by oncogenic mutation  continuous downstream signalling GAP has almost no effect on mutant ras oncoproteins

What does Ras do?       

Binds to GDP in inactive state Releases bound GDP on receiving stimulatory signal from upstream signalling cascade (GRB2 and SOS) Acquires GTP in place of GDP Shift into activated, signal-emitting configuration when bound to GTP Enables Ras to bind to Raf  recruit to plasma membrane  activates Raf Cleave this GTP after short period Return to inactive state

Oncoproteins: signal is not turned off - Emits signal for long time (indefinite) - Flooding cell with these signals The ras proto-oncogene encodes a protein of 189 amino acids. In the normal protein, glycine is encoded at position 12 and glutamate at position 61. Analysis of ras oncogene proteins from several tumours shows a single amino acid substitution at one of these positions, which converts a protooncogene into a tumour-promoting oncogenes. Oncoproteins due to missense mutations Changes at position 12, 13 or 61 Located in cavity in Ras for GTPase catalytic activity (hydrolysis) Oncogenic Ras is to drive cell proliferation and transform a cell

MAPKinase Pathway Cascade of protein kinases highly conserved  central role in signal transduction in all eukaryotes from yeast to humans Activated Ras activates signal transducer RAF. RAF phosphorylates and activates MEK. MEK phosphorylates and activates MAPK. Activated MAPK enters the nucleus and phosphorylates and activates transcription factors that turn on specific sets of genes needed for cell growth

Oncogenes and the ERK pathway Oncogene proteins act as growth factors (EGF), growth factor receptors (ErbB), and intracellular signalling molecules (Ras, Raf and MEK). Ras, Raf and MEK activate the ERK MAP kinase, leading to the induction of additional genes (fos) that encode potentially oncogenic transcriptional regulatory proteins. EGF, ErbB, Ras, Raf, MEK and fos mRNA are proteins with known oncogenic potential.

AP-1 transcription factor AP-1 transcription factor (dimer of Fos and Jun)  activates transcription of cyclin D1 and other growth factor-inducible genes AP-1 binds to DNA  regulates expression of genes involved in

 Growth  Differentiation  Death Cyclin D and Cdk  passes checkpoint in cell cycle

Gatekeeper vs caretaker genes  

Gatekeeper genes  control the cell cycle; mutation ‘opens the gate’ to unregulated cell division Caretaker genes  maintain genomic integrity; DNA repair genes, repair DNA damage and prevent genomic instability

Lecture 3 Retinoblastoma Tumour suppressor genes Experimental evidence in the 1970s and 1980 – - Hinted at the existence of a second, fundamentally different type of growth controlling gene  one that operates to constrain or suppress cell proliferation. - The antigrowth genes came to be called tumour suppressor genes  Their involvement in tumour formation seemed to happen when these genes were inactivated or lost.  Inactivation of tumour suppressor genes plays a role in cancer pathogenesis that is an important to cancer as the activation of oncogenes  When DNA of cancer cells and normal cells combine  hybrid cell is either tumorigenic so cancer alleles are dominant or hybrid cell is non-tumorigenic so cancer alleles are recessive

Tumour suppressor genes: Function (breaks)     

Gene products halt progression through cell cycle Prevent mitotic division For cell division: these products must be inactive or absent If permanent inactivation or mutation of tumour suppressor gene  loss of cell cycle control Uncontrolled proliferation

Retinoblastoma Rare inherited cancer syndrome in children RB1 gene  retinoblastoma-associated protein (pRb or Rb) Malignant tumour of retina of eye in young children  Rare  1 in 18-20000  Presents: 1-3 years  Retinoblasts  Tumour originates in embryonic retinal cells: until age 2 Mature retinal cells do not transform into tumours  why tumour usually occurs only in children Loss of Rb function leads to increase in cell number, failure of differentiation and tumour formation - Thickening of optic nerve due to extension of tumour - Displaced normal retina - Retinoblastoma Initially a retinoblastoma may present at clinic as an opacity that obscures the retina  Loss of vision  Fatal if not treated  Laser and cryotherapy for early tumours

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Chemotherapy, radiotherapy and enucleation of the eye for advanced disease Best results with early diagnosis

Hereditary retinoblastoma    

Inheritance of predisposition associated with loss of chromosome 13q14 Autosomal dominant trait  over 90% will develop retinal tumours: high penetrance Development of cancer: recessive at cellular level Loss of both alleles

Chromosomal localization of Rb locus Karyotype of retinoblastoma of a 6 year old patient  interstitial deletion of long arm of chromosome 13 Clinical course of 1601 retinoblastoma patients: those with bilateral disease have a much higher risk of subsequently developing cancer in a variety of organ sites than those with unilateral disease Pedigree of familial retinoblastoma Children can be cured of the disease and can reproduce Arrows on pedigrees shows an example of incomplete penetrance Alfred Knudson Studied 2 types of retinoblastoma Sporadic  Both RB1 alleles must mutate in same retinal cell for tumour to develop  Less frequent event; one eye; later age  Two independent spontaneous somatic mutations in same retinal cell Familial  Germ line mutation in one RB1 allele  In all cells in body  Including retinal cells  If 2nd allele mutates in retinal cell – tumour  15% cases  no 2nd mutation  no cancer  One i...