Cancer Biology - Lecture notes All PDF

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Introduction to Cancer Biology 1 Tumours/neoplasms (‘new growth’) - arise from normal tissues, tumour cells lose form and function, often have large nuclei Divided into benign (non-invasive, localised, retains some normal form and function) and malignant (invasive, metastatic, can be traced back to ...

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Introduction to Cancer Biology 1 Tumours/neoplasms (‘new growth’) - arise from normal tissues, tumour cells lose form and function, often have large nuclei Divided into benign (non-invasive, localised, retains some normal form and function) and malignant (invasive, metastatic, can be traced back to primary tumour) Primary tumour - original site, metastases known as secondary tumour, but are same cell type as original Classified into 4 main groups based on cell type of origin - epithelial, mesenchymal, haematopoietic, neuroendodermal Epithelial cells line walls/cavities or form outer covering (skin, cornea, bronchiole), also secrete proteins to form basement membrane (ECM, proteins) attached by collagen to support tissue Epithelial tumours - 80% of known tumours Carcinomas - squamous cell carcinomas (protective layer epithelial cells) eg skin, nasal cavity, lung, esophagus; adenocarcinomas (secretory epithelial cells) eg lung, colon, breast, pancreas Other types: small-cell lung carcinoma (common in smokers, thought cancers arise from neuroendodermal cells), large-cell lung carcinoma, renal cell carcinoma Mesenchymal tumours - derived from connective tissue cell types (originally from embryonic mesoderm), ~1% tumours Sarcomas - eg osteosarcoma (bone), liposarcoma (adipocyte), fibrosarcoma (fibroblast), angiosarcoma (endothelial cells, lining of blood vessels) Haematopoietic tumors - derived from cell types in ‘blood forming’ tissues and immune system, ~7% tumours Leukemia - malignant cell derivatives that move freely in circulation Lymphoma - solid tumours of B or T lymphocytes, commonly found in lymph nodes Neuroendodermal tumours - ~1.5% tumours, derived from components of CNS and PNS eg glioblastomas, neuroblastomas, astrocytomas Some tumours do not fit into the four classifications eg melanoma derived from skin melanocytes Cancers appear to develop progressively with tumours demonstrating different gradations of abnormality Benign tumours: Hyperplastic - excessive cell number, cells are ‘normal’ and form structure Metaplastic - displacement of normal cells with other normal cells not normally found in that tissue, frequently found in epithelial transition zone, can be premalignant eg Barrett’s oesophagus - normal squamous cells of lower oesophagus replaced by secretory cells of the stomach, promoted by chronic acid reflux - 30% increased chance of developing oesophageal carcinoma Dysplastic tissue (not associated with increased proliferation): Cytologically abnormal cells - variable shape and tightly packed, hyperchromatic nuclei (takes up more stain), lack differentiation markers (hallmark of premalignancy) Malignant tumours: cells breach basement membrane and invade surrounding stroma, abnormal cells with poor differentiation, spread to distant sites

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Tumours are monoclonal growths - currently debated Derived from a single abnormal cell Multiple myeloma - tumours derived from B cell precursors, myelomas cells demonstrate a ‘spike’ of single antibody species, indicating the tumour is derived from a single B cell clone (visualised with gel electrophoresis) Cancer cells show altered glucose metabolism Normal cell - import glucose, fed into glycoylsis - generate 2ATP, pyruvate kinase M1 forms pyruvate and ensures pyruvate transported into mitochondria → acetyl CoA → Krebs cycle → electron transport chain → 32 ATP Cancer cells have pyruvate kinase M2 - pyruvate becomes susbtrate for lactate dehydrogenase and lactate is produced - tumour cells actively promote this aerobic glycolysis ‘Warburg effect’ to generate their ATP even in ample oxygen levels Common oncogenes promote the processes involved in aerobic glycolysis - glucose uptake and lactate generation Cancer cells utilise aerobic glycolysis - frequently hypoxic (lack oxygen for oxidative phosphorylation, process of glycolysis also generates intermediates for biosynthetic pathways such as growth, tumour growth depends on expression of pyruvate kinase M2, GLUT1 and lactate dehydrogenase

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If cancer was mainly due to random DNA mutations, cancer incidence would increase regularly with increased number of cell divisions - but there are large variations between countries - overwhelmingly environment and dietary choices are responsible for the variation Migrating populations - lowest rate of prostate cancer in Asian men, but upon migrating their prostate cancer incidence matches the Caucasian population of where they have migrated to Both physical and chemical carcinogenic agents act as mutagens - mutagenicity measured by the Ames test Not all carcinogens mutate DNA eg tumour promoters (stimulate processes involved in cell proliferation and growth) Tobacco-related cancers - 34% total cancers in US High fat diet - 37% Screening - detect in early stages before spread beyond local site, before symptoms appear, easier and cheaper to treat, treatment more likely to be successful 3

WHO criteria for screening: condition, test, treatment, screening programme Condition - must be important health problem, requires understanding of natural history of the disease (opportunity to interfere with disease process, needs factor detectable at early stage), must be costeffective to prevent rather than treat (treating metastatic tumours is extremely expensive), requires understanding of implications of detecting people with disease Test - simple, safe, precise, validated, reliable, easy and safe to administer, marker easy to measure (eg in blood, urine), have agreed and suitable cut-off for positive/negative results, need to determine specificity and sensitivity, not invasive or time consuming (acceptable to population), clear procedure for follow-up of positive test (further tests, treatment) Treatment - must have effective treatment established, should be improved outcome compared to diagnosis at later stage of disease, clear procedures for treating and managing patients Screening programme - must have been effectively trialled, shown to reduce morbidity (incidence) and/or mortality (number of deaths), benefits must outweigh the physical and psychological harm of test, patients must be fully informed of consequences of being tested, economically effective and viable UK screening programmes: Colorectal cancer - tests foetal occult blood, age 60-74 Cervical cancer - age 25-65 Breast cancer - mammography, 50-70 Most invading cancers are detected at stage 4, those detected earlier are often done so by accident as a result of another test Introduction to Cancer Biology 2 Genetic diseases arise due to mutations in the genome, could be inherited - passed from generation to generation via the germline ‘heritable’, could be somatic - arise after conception in non-germ line cells (unique to the individual) these are the minority Cancer is a disease of cells - loss of normal cell behaviour, cell number tightly regulated by proliferation, apoptosis, differentiation, processes regulated by genes or products of genes Regulation of gene activity - multiple mechanisms of transcriptional, translational and posttranslational regulation, also epigenetic regulation which affect the promoters Most carcinogens are mutagens - damage DNA: chemical carcinogens, radiation, infectious pathogens eg HPV, natural cell processes (ROS, replication errors) ● Pecorino Chapter 2 CC to TT transition mutations are specific to UVB induced mutations - unique to UV damage and prevalent in skin cancer Increasing the rate at which DNA is damaged increases the chance of altering a gene that controls proliferation/apoptosis Types of mutation: point: transition (purine-purine, pyrimidine-pyrimidine) or transversion (purinepyrimidine), deletion, insertion, duplication, inversion, translocation, aneuploidy (latter two more associated with the later stages of cancer, smaller mutations associated with early cancers) Estimated 104 to 106 mutations occur per cell per day 4

Type of mutation can be associated with cancer aetiology Eg UV light - most commonly seen in melanomas and head and neck cancers, smoking more common in lung, head and neck cancers, BRCA1/2 associated with breast, ovary and pancreas Carcinogenesis is a multi-step process driven by accumulation of mutations (not necessarily a linear process), tumours are known to possess multiple mutations - not homogeneous Eg FAP (familial adenomatous polyposis - inherited) - Vogelstein developed adenoma to carcinoma sequence: Early stage adenomas (no invasion of lymph nodes/surrounding tissue) - 95% survivable Acquire new mutations, spread to lymph nodes and metastasise around body - poor prognosis - 33% survivable beyond 5 years Number of genes mutated increases with later stages Pathophysiology - lose distinct villi, hyperproliferation of intestinal epithelia Adenomatous polyposis coli (APC) - tumour suppressor gene Tumour suppressor gene - growth inhibitors APC - must lose both copies of gene before seeing disease phenotype (recessive) Homozygous APC mutation is embryonic lethal - critical in Wnt signalling axis patterning Familial FAP - inherit heterozygous APC mutation, loss of heterozygosity occurs after birth somatic mutation of second allele occurs in intestine → hyperproliferative epithelia → benign adenomas Somatic mutation does not matter in cells in which the gene is not crucial to the cell’s function Sporadic - (majority) separate somatic mutations in both alleles *retinoblastoma mutations in Pecorino - lookup K-Ras, B-Raf mutations can convert hyperproliferative epithelia → adenoma Ras and Raf are proto-oncogenes Proto-oncogenes - normal genes that have functional roles in normal cells, drive growth, can be activated by mutation to be oncogenic - too much protein is produced eg growth factors, or gene may acquire mutation preventing posttranslational modifications and the function is always ‘switched on’ Often proto-oncogene mutations are dominant - only one allele is sufficient to drive tumorigenesis Differences between tumours is inter-tumour heterogeneity

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Tamoxifen - blocks function of oestrogen receptor Herceptin - Mab, binds to HER2 receptor on tumour and blocks it, tumour must be HER2 positive to respond, ‘personalised medicine’ Differences within tumours is intra-tumour heterogeneity Current view - cancer begins from a single cell, additional different mutations are acquired as the cancer progresses and grows further Selective pressures drive evolution of cancer Evolution can be mapped through genome sequencing, to see accumulation of genes mutated that gives cells the selective advantage to either stay in the current tumour or to metastasise B-Raf - serine threonine kinase - phosphorylates other proteins, mutated in melanoma, specific activating mutation can be targeted by small molecule inhibitor B-Raf phosphorylates MEK1 (now active) but if MEK1 has mutation preventing it from being affected by B-Raf, it may no longer be affected by the drug targeting B-Raf - tumour recurrence Tracking cancer evolution in blood - liquid/virtual biopsy - isolate circulating tumour DNA in plasma/those that have been released into circulation after death - whole genome sequence to understand mutation profile - can analyse acquired resistance to cancer therapy, tracking of disease progression and response to therapy, potential for use in clinical diagnosis and screening P53 - guardian of the genome - very important tumour suppressor gene, associated with adenoma → carcinoma transition Chromosomal aberrations associated with metastasis P53 activated by DNA damage, aberrant growth signals, oncogene activation, cell stress (hypoxia, nucleotide depletion) Once activated can cause cell cycle arrest, apoptosis, DNA repair, inhibition of angiogenesis 6

P53 Haploinsufficiency - losing one allele copy (lower gene dose) is sufficient to result in abnormal growth - can reduce DNA repair proteins DNA damage repair - base excision repair, nucleotide excision repair, homologous recombination etc DNA repair genes - mutations mean unable to repair DNA Nucleotide excision repair - PARP recognises single stranded breaks, binds and initiates other protein binding and enzymatic repair BRCA - mutation in 1/2 cause ~20% inherited breast cancers but only ~2% of all breast cancers, involved in DNA repair - homologous recombination for double stranded breaks, uses sister strand as template

Synthetic lethality - exclusively kills BRCA2 mutant cells PARP inhibition - single strand break not repaired - progresses to double stranded break BRCA and PARP inhibition - no DNA repair

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Treat breast cancer patient who is BRCA mutant with PARP inhibitor - only BRCA deficient cells should die as they cannot repair DNA using either mechanism 5-10% of all cancers arise from inheriting a mutation which predisposes to an increased risk *youtube synthetic lethality Cell Cycle and Cancer

Mouse - 40 chromosomes Lee Hartwell discovered cdc proteins - worked with budding yeast - can tell where they are in cell cycle by bud size

Used to find mutations - nitrosoguanidine as mutagen to look for temp sensitive mutations that alter cell cycle kinetics - will all be lethal to cell, need mechanism to hide mutations until needed, therefore temp sensitive to change conformation of protein

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Catalogued mutations as to where they stopped yeast progressing through cell cycle Cdc28 - blocks passage through any part of cell cycle - mediates early event necessary for cell cycle to start transition from G1-S Paul Nurse - Cdc2/Cdk1 using budding and fission yeast Fission yeast extends, wall forms in the middle and division occurs - use size to determine position in cell cycle Mutation reducing cycle duration - smaller cell generated Increasing cycle duration - bigger daughter cells Cdc2 controls transition from G2 to M in fission yeast and has same function as Cdc28 in budding yeast Cdc2 controls both G1→ S and G2 → M Corresponding human gene Cdk1 - encodes protein that is member of cyclin dependent kinase family add phosphate groups James Maller - maturation promotion factor identified in frogs, extract of mature oocytes implanted into immature oocytes → initiated capacity to divide, also key regulator of general cell cycle Tim Hunt - discovery of cyclins, proteins specified by maternal mRNA in sea urchin eggs, is destroyed at each cleavage division, he coordinated expression changes following fertilisation Cyclin A protein levels oscillate, falling at onset of cleavage, variations in intensity of cyclin due to destruction by periodic proteolysis, not to periodic synthesis Emryologists purified MPF and found it consisted of two subunits: cdc2 and Cyclin B Cdk and cyclin together form an enzyme that activates other proteins by phosphorylation, the amount of Cdk molecules is constant during the cell cycle, but their activities vary because of the regulatory function of the cyclins Cdk acts like the engine, cyclin like a gear box controlling whether the engine will be idle or drive the cell forward in the cell cycle

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Only Cdk1 is essential for cell division - mutation results in embryonic lethality in first cell divisions Cdk2-/- sterility due to defective meiosis, no effect on mitotic cells Cdk4-/- diabetes Cdk6-/- slight anaemia, defective hematopoietic proliferation Cdk2-/-, Cdk4-/-, Cdk6-/- embryonic lethality by mid-gestation due to hematopoietic defects First tumour suppressor genes identified are cell cycle regulators At S phase, cyclin D/Cdk4/Cdk6 phosphorylate pRb (retinoblastoma), inhibiting its activity, further phosphorylated by cyclin E/Cdk2 pRb remains phosphorylated throughout S, G2 and M Rb binds and inhibits TFs of the E2F family, which prevents G1→ S transition

Bi-allelic Rb loss directly leads to retinoblastoma, near 100% penetrance, only known tumour driven by a single gene mutation P16 - Cdk inhibitor, presence associated with senescence, loss associated with malignant disease

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These proteins effectively mediate checkpoints

Chromosome separation monitored by anaphase promoting complex regulating cyclin A and cyclin B Cyclin B Cdk1 suppresses separase and keeps chromatids together If Cdk1 rendered inactive by APC - chromatids separate If this machinery does not work correctly - leads to genome instability Cdh1 stabilises targets allowing separase to work Visualise chromosomes by painting each a different colour with fluorescence Cancer cells can display fluorescence of different colours across each chromosome, various sizes and number of copies Checkpoints permit cell cycle arrest to allow repair of damaged DNA/correct chromosome segregation and activation of a death programme - apoptosis (safe removal of structures that are deemed dangerous/unnecessary eg webbing between fingers)

Checkpoint failure leads to tumorigenesis p53 and Rb - key to development of invasive, aggressive cancer, elevated in majority of cancer types Range of drugs currently being trialled that target Cdk to suppress cell cycle in cancer cells 11

Tumour terminology: Hyperplasia - an increase in the number of normal cells in a tissue or organ Dysplasia - abnormal development or growth of tissues, organs or cells Cancer in situ - involves only the place in which the cancer began and has not spread, carcinoma in situ is an early-stage tumour Invasive cancer - begins in one area and spreads deeper into tissues of that area Metastatic cancer - spread from the place it began into other tissues and parts of the body - moved via blood vessel and built new niche Oncogenes - genes which, if mutated, can drive tumorigenesis eg H-Ras Tumour suppressors - genes which normally work to suppress tumorigenesis eg p53, Rb Gain of function - mutations which give/enhance function, particularly in oncogenes Loss of function - inactivating mutations, particularly in tumour suppressors Breast, lung, colorectum and prostate cancer have highest incidence in UK Males aged 70+ will normally have some form of prostate cancer, may be benign Malignant melanoma - incidence increased ~70% in last 5 years, associated with sun exposure Bladder cancer - incidence decreased, associated with chemicals inhaled in workplace Melanoma ~90% 5 year survival rate (for early diagnosis, not malignant melanomas) Hodgkin’s lymphoma ~85% Lung ~6% Pancreatic ~2% Epigenetics and Cancer Mutating a gene is not the only way to switch it on/off as the expression of all genes is regulated by chromatin architecture - epigenetics

All cells in an organism are genetically equivalent - the epigenetic landscape determines cell fate Waddington - developmental fate determined by epigenetics, can be applied to cell fate in tumorigenesis Epigenetics - heritable changes in gene expression that occur without alteration in DNA sequence Primary epigenetic mechanisms - DNA methylation and covalent modification of histones 12

RNA is intimately involved in the formation of a repressive chromatin state eg X inactivation DNA methylation occurs almost exclusively at CpG dinucleotides, most CpG sequences in the genome are methylated, methylation is catalysed by family of DNA methyltransferases (DNMTs) De novo methyltransferases newly methylate cytosines, expressed mainly in early embryonic development, set up the pattern of methylation, primarily DNMT3a and 3b DNMT3L is implicated in imprinting Maintenance methyltransferases add methylation to DNA when one strand is already methylated, work throughout life of organism to maintain methylation, primarily DNMT1 CpG island hypermethylation at promoter seen in colon (p16), breast (BRCA1), lung cancer etc Global hypomethylation seen in colon, bladder Genomic DNA methylation levels are delicately balanced within cells, overexpression of DNMTs is linked to cancer, their deletion is lethal Methylcytosine (CpG with methyl group) is capable of spontaneously mutating in vivo by deamination to give thymine, responsible for 37% of somatic p53 gene mutations and 58% of germline mutations Hypermethylation of CpG islands at tumour suppressor genes switches them off Global hypomethylation leads to genome instability and inappropriate activation of oncogenes and transposable elements Normal pattern of methylation -...