Cancer Pathology Lecs (AutoRecovered) PDF

SEMESTER 2 2024 DEF IN IT IO NS Neoplasm – “an abnormal mass of tissue, the growth of which exceeds and is uncoordinated with that of normal tissues, and persists in the same excessive manner after apparent cessation of the stimuli which evoked the change” Neoplasm (tu...

SEMESTER 2 2024 DEF IN IT IO NS Neoplasm – “an abnormal mass of tissue, the growth of which exceeds and is uncoordinated with that of normal tissues, and persists in the same excessive manner after apparent cessation of the stimuli which evoked the change” Neoplasm (tumour) – an abnormal mass of tissue that proliferates rapidly and in an uncontrolled way. o continuous increase in the number of dividing cells. o Neo=new, plasm= thing formed Hyperplasia Hypertrophy Dysplasia Neoplasia NEO PL AS M All have 2 basic components: o CLONAL CELLS from one type of “progenitor” ▪ range from mature to totally immature primitive cells mature – recapitulate normal tissue to a degree o REACTIVE STROMA made up of connective tissue, blood vessels, and inflammatory cells CLA SS IF IC AT IO N O F TU MO UR S Histo-genetic (morphology, molecular) Tumours with a o site of origin – classifying where tumour comes from high mutational o behavioral – benign/malignant load do better prognostically Aetiological – environmental factors? Therapeutic biomarkers - molecular studies CLA SS IF IC AT IO N B Y BI OL OG IC AL B EHA VI OU R Benign – will remain localized, can be completely excised and wont return o E.g. naevus (local melanocytic proliferation that is benign and wont spread but may grow a little bit), colon adenoma Malignant – invasion (locally or spread) o E.g. melanoma, colonic carcinoma Intermediate/Borderline – risk of recurrence o E.g. GIST, pheochromocytoma CLASSIFICATION BY HISTOGENESIS Epithelium – skin or mucosal o Adenoma – a gland forming epithelial tumour o Carcinoma – a malignant gland forming epithelial tumour Mesenchyme – stroma/connective tissue elements o Leiomyoma o Leiomyosarcoma ▪ “Leio” – smooth muscle, leiomyoma – benign, smooth muscle tumour, leiomyosarcoma – malignant, smooth muscle tumour o Solitary fibrous tumour Blood and related cells o Leukaemia Special cells – melanocytes, germ cells o Naevus, melanoma, seminoma Mixed NOMENCLATURE: GENERAL RULES Most neoplasms have suffix -oma Tissue type of origin + oma = benign o E.g. lipoma, chondroma, adenoma, osteoma Cell/tissue type + carcinoma = malignant o E.g. adenocarcinoma (adeno: relating to glands), urothelial carcinoma, squamous cell carcinoma Cell/tissue type + sarcoma = malignant o E.g. chondrosarcoma, liposarcoma, osteosarcoma EXAMPLE LESIONS ARISING FROM COLONIC MUCOSA Adenoma = benign neoplasm o A tumour which is clonal without capacity to invade Adenocarcinoma = malignant neoplasm o A tumour having the properties of invasiveness and/or metastasis COLECTOMY DUE TO HISTORY OF FAMILIAL ADENOMATOUS POLYPOSIS - Polyps are neoplastic E.g. Colorectal adenoma No invasion Still benign but may have some growth E.G. LOW GRADE DYSPLASIA (ADENOMA) By definition, if the tumour growth has not infiltrated the muscularis mucosa, it's still a benign lesion because it's sitting within the mucosa propria E.G. COLONIC ADENOCARCINOMA Malignant From a colectomy Malignancy has gone into muscularis propria METASTATIC COLONIC ADENOCARCINOMA - Liver  EXAMPLE SKIN LESIONS Similar clinical appearance may have a very different prognosis Ulcerated lesion with a raised white edge to it. That could potentially even be a malignancy. Although the raised white edge would indicate there's probably a growth there. SQUAMOUS CELL CARCINOMA Infiltrating between collagen bundles BASAL CELL CARCINOMA Less eosinophilic cytoplasm Peripheral palisading EXAMPLE SARCOMAS Osteosarcoma Leiomyosarcoma Liposarcoma (background mineralised bone) (smooth muscle fibres) (adipocyte-like cells) CLINICAL PRESENTATION OF NEOPLASIA Primary vs Secondary (metastasis) Requires correlation with previous diagnoses, imaging, morphology, immunohistochemical profile By the time you get symptoms the tumour may have spread Primary – comes from that site Secondary – infiltrated or metastasis from somewhere else DIFFERENTIATION = The extent to which neoplastic parenchymal cells resemble the corresponding normal cells, both morphologically and functionally Anaplasia = lack of differentiation / undifferentiated o (ana= backward, plasia= formation) MORPHOLOGICAL CHANGES ASSOCIATED WITH ANAPLASIA Pleomorphism – variation in size and shape Mitoses – atypical, bizarre mitotic features, sometimes producing tripolar or multipolar spindles o Reflect higher proliferative activity of parenchymal cells Loss of polarity – anaplastic cells grow in a disorganized fashion Abnormal nuclear morphology: o Disproportionally large for the cell o Nuclear-to-cytoplasm ratio may approach 1:1 instead of normal 1:4 or 1:6 o Shape is variable and often irregular o Chromatin is often coarsely clumped and distributed along the nuclear membrane o Usually contain large nucleoli  “Just about any tumour type shows a degree of backwards differentiation in that they become more immature looking than a normal cell of that cell type. Anaplasia tend to anaplastic cells and tend to show a lot of pleomorphism so difference in cell size and shape. So the cells have become undifferentiated” Anaplastic tumour of the skeletal muscle (rhabdomyosarcoma). Note the marked cellular and nuclear pleomorphism, hyperchromatic nuclei, and tumour giant cells. METAPLASIA Precedes neoplasia The replacement of one type of cell with another type o Usually in association with tissue damage, repair and regeneration Normally squamous cells line the oesophagus In the process of metaplasia, these are replaced by columnar intestinal- type cells Metaplasia here = Barrett oesophagus Precursor lesion (= risk of) oesophageal carcinoma DYSPLASIA Disordered growth, often occurs in background metaplastic epithelium Architecture of the epithelium becomes disorderly Often found adjacent to foci of invasive carcinoma Dysplasia does not necessarily progress to cancer Once the tumour cells breach the basement membrane, the tumour is said to be invasive High grade dysplasia – carcinoma in situ (most grading systems) “Dysplasia = neoplasia that is still confined to the site at which it's occurring. So, within, in this case, the basement membrane in a colorectal polyp or a colorectal adenoma to be more precise, confined by the muscularis mucosa. GRADING BY DIFFERENTIATION Different tumours have different grading BREAST CANCER – NOTTINGHAM GRADING SYSTEM Grades breast tumours based on: o Tubule formation – how much of the tumour tissue has normal breast duct structures o Nuclear grade – an evaluation of the size and shape of the nucleus in tumour cells o Mitotic rate – how many dividing cells are present (a measure of how fast the tumour cells are growing and dividing) Each of the categories gets a score between 1 and 3 o Score of 1 = cells and tumour tissue look the most like normal cells and tissue o Score of 3 = cells and tissue look the most abnormal Scores for the three categories are added, yielding a total score of 3 -9 Higher grade = spread more likely Grade 1 Total score = 3-5 Low grade or well differentiated Grade 2 Total score = 6-7 Intermediate grade or moderately differentiated Grade 3 Total score = 8-9 High grade or poorly differentiated PROSTATE CANCER: GLEASON SCORING SYSTEM Biopsy taken from the prostate Both a primary and a secondary pattern of tissue organization are identified o Primary pattern represents the most common tissue pattern seen in the tumour o Secondary pattern represents the next most common pattern Each pattern is given a grade from 1-5 o 1- most like normal prostate tissue o 5- most abnormal The two grades are then added to give a Gleason score Gleason scores are grouped into the following categories o Gleason X – Gleason score cannot be determined o Gleason 2-6 – the tumour tissue is well differentiated o Gleason 7 – the tumour tissue is poorly differentiated or undifferentiated By the time a solid tumor is clinically detected, it has often completed a major portion of its life span. Major impediment in the treatment of cancer and underscores the need to develop diagnostic markers to detect early cancers (or less toxic systemic treatments) FORMATION OF METASTASES Penetrate the basement membranes/capsules Movement through extracellular matrix Penetration of vascular channels Survival/arrest in the circulation Exit to new tissue sites Survival and growth as metastasis evoking angiogenesis (the growth of blood vessels from the existing vasculature – feeds tumour)  Endovascular junctions are less COMMON SITES FOR METASTASIS intact And so livers are sort of a Bone site where cells can potentially get Liver out and again in the bone marrow is Brain where hematopoietic elements are Lung – blood flow coming into the circulation and Lymph nodes – lymph goes through organs coming out of the circulation so and back to vascular systems bone stroma is a site where tumours seen SPREAD OF CARCINOMA Local direct invasion Intraepithelial spread Lymphatic dissemination Hematogenous dissemination Spread across body cavities o Pleural, peritoneal, cerebrospinal Others – iatrogenic spreads - induced unintentionally by a physician or surgeon or by medical treatment or diagnostic procedures o Implantation, fine needle aspiration tract (sarcomas spread commonly this way) EFFECTS OF METASTASIS Destructive growth in vital organs – liver, lung, brain etc. is an important cause of death Responsible for significant morbidity as well as mortality – e.g. bone metastases → pain, disability, fractures… CANCER STAGING = Degree of tumour spread Assessment of likely prognosis, directs treatment TNM staging o T – Tumour : size, local spread o N – Nodal status : number, groups, size etc (Lymph node) o M – Metastasis Staging has a decision on chemotherapy, suggests prognosis Aetiology – the study of the cause of something Carcinogenesis aka oncogenesis or tumorigenesis – the process by which normal, healthy cells transform into cancer cells LECTURE OBJECTIVES 1. DESCRIBE THE MULTI-STEP PROCESS OF CARCINOGENESIS HALLMARKS OF CANCER – SOMETHING FOR EVERYONE MULTI-STEP PROCESS OF MUTATION ONCOGENES & TUMOUR SUPPRESSOR GENES 2. APPRECIATE THE TYPES OF CARCINOGENS THAT HAVE BEEN PROVEN TO CAUSE CANCER AND DESCRIBE HOW THEY DO THIS CARCINOGENESIS FIRST STEPS: ENDOGENOUS VS EXOGENOUS ENVIRONMENTAL CARCINOGENS – MECHANISMS OF ACTION CANCER MUTATIONAL BURDEN MUTATIONAL SIGNATURES MULTI-STEP PROCESS OF MUTATION Cancer is ultimately a disease of tissue growth dysregulation and uncontrolled proliferation – but how does it start? Somatic mutation theory – cancers arise from mutations in individual cells, passed on through division (endogenous or exogenous cause) Clinically observable cancer is the result of accumulating MULTIPLE mutations Given that normal cells are very good at fixing errors in DNA, how might multiple mutations occur?  Mutator phenotype – an acquired increase in rate of mutation o First ‘mutator’ acquisition will often relate to: ▪ Increased cell division, or; ▪ Abnormal DNA replication, or; ▪ Damaged DNA repair mechanisms Mutations can have following consequences: o LETHAL - Disruptive in a manner that harms or kills the cell; o PASSENGER - Consequential, with no selective advantage; o DRIVER - Provides a selective advantage (LEAST COMMON). A study in colon cancer found that an average of 15 driver & 60 passenger mutations per tumour. Mutations to various classes of genes are normally present, but mutation is random so: o No predetermined order; o Myriad mechanisms altered along the path of tumorigenesis; o Co-opting existing cellular pathways - Cancer-causing mutations are cumulative but randomly ordered Mutation can occur in any cell at any time Most likely to occur during DNA replication Will only be passed on if cell divides (clonal expansion) Stepwise mutations result in heterogeneity: o Between tumours in an individual with metastatic disease o Within a single tumour Tumour heterogeneity has implications for therapy  Tumours co-opt existing cellular pathways For most of the ‘hallmarks of cancer’ there are associated networks of genes that can be disrupted Examples: o VEGF disruption can change angiogenesis o P53 disruption can interfere with apoptosis o MAPK signaling can impact on tissue invasion, growth signals self- sufficiency AND proliferation ‘natural selection’ in miniature – many selective pressures e.g.: o Immune system o Tumour suppressor genes o Microenvironment e.g. anaerobic respiration under hypoxia FEEDBACK LOOP LESS tumour cell death MORE proliferation MORE rounds of DNA replication MORE chance of passing mutations on MORE opportunity for mutation and instability ONCOGENES & TUMOUR SUPPRESSOR GENES ‘Pushing the accelerator’ vs ‘releasing the brakes’ ONCOGENES Proto-oncogenes typically are one of: o Cell division stimulators; o Differentiation blockers; o Apoptosis inhibitors; o Components of signaling pathways; o Growth factors. Proto-oncogenes become oncogenes when expressed at increased levels, resulting from either: o Amplification leading to more copies of the gene; o Translocation to a more active promotor o Mutation resulting in a fusion protein with oncogene activity >40 oncogenes currently known ONCOGENES – MYC Transcription factor Overexpressed/activated in >50% of human cancers Coordinates many cellular processes Induces ‘stemness’ Blocks senescence Blocks differentiation ‘MYC addiction’ – tumour survival often depends on high MYC Not sufficient alone for carcinogenesis ONCOGENES – RAS GTPase signalling proteins 3 RAS genes – KRAS, NRAS, HRA RAS mutants are 85% KRAS Active (phospho) RAS then activates downstream GTPases Signalling cascades RAS on/off activity is sped up by regulatory proteins: o Guanine nucleotide exchange factors (GEF) catalyse exchange of GDP with GTP (RAS “switched on”) o GTPase-activating proteins (GAP) catalyse hydrolysis of GTP to GDP (RAS “switched off”). Mutant RAS don’t allow GAP to coordinate hydrolysis - RAS is stuck ‘on’. TUMOUR SUPPRESSOR GENES (brief – covered in other lectures) “anti-oncogenes” Problem if these are knocked out: examples in figure → Often requires “two-hit” effect i.e. both alleles K.O. (opposed to oncogenes needing “one-hit” as gain-of function) Hereditary susceptibility, e.g. if one allele is already affected then only the existing ‘good’ one needs to be knocked out Often have suppressive or regulatory activity e.g.: o Control proliferation o Initiate apoptosis if DNA damage is detected o Regulate adhesion i.e. stop metastasis HOW DO WE ACQUIRE MUTATIONS? What type of mutations can occur? o LARGE SCALE (less common): ▪ Usually happen during mitosis or meiosis Chromosomal gain/loss (usually lethal) o Down syndrome (trisomy 21) and Klinefelter syndrome (XXY) both associated with leukaemia Translocation/duplication/deletion of large fragments o ‘Philadelphia chromosome’ (translocation between long arms of chromosomes 9 and 22) seen in 85% of patients with chronic myeloid leukaemia. o D deletion syndrome (loss of long arm of chromosome 13) associated with retinoblastoma o SMALL SCALE (more common): ▪ Point mutations ▪ Proteins can still be made ▪ Very subtle differences ▪ May prevent one particular role i.e. binding to a specific partner ▪ Frame shift in ORF – protein not made CARCINOGENESIS: FIRST STEPS ENDOGENOUS CAUSES Hereditary (3,200 annual cases of cancer in Australia attributable to long-term alcohol consumption (2010 estimate). Increases risk of mouth, pharynx, larynx, oesophagus, bowel, liver, breast and probably stomach cancer. Via blood stream, can affect many tissues/organs around the body. Mechanisms: o 1) Ethanol -> acetaldehyde by alcohol dehydrogenase - damages DNA & stops cells from repairing; o 2) Ethanol may also cause direct tissue damage to cells of mouth/throat; o 3) Acts as a solvent for other carcinogens (e.g., from smoking); o 4) Increases level of hormones such as oestrogen (linked to breast cancer) or insulin EXAMPLE 2 – RADIOLOGICAL: UV RADIATION Skin cancer is most common cancer in Australia. >13,000 new cases & >1,700 deaths annually (2016) 99% of non-melanoma skin cancers and 95% of melanoma caused by UV Sources – sun, solariums, sunbeds, and sun lamps UVA penetrates into dermis. Genetic damage to cells, photo-ageing, immune- suppression. UVB penetrates into the epidermis. Damages cells, responsible for sunburn -> melanoma If damage isn’t repaired, cell may grow in an uncontrolled way.  Researchers examined eyelid skin from 4 people undergoing cosmetic surgery None had ever had skin cancer A quarter of all cells had mutations associated with squamous cell carcinoma Many ‘pre-cancerous’ cells were growing as clones (i.e. in clusters) High frequency contained driver mutations (some with 2 or 3) Why no cancer then? IMMUNITY… covered elsewhere EXAMPLE 3 – BIOLOGICAL: HUMAN PAPILLOMAVIRUS (HPV) Globally common viruses >100 types, ~14 can cause cancer Two types (16 & 18) cause 70% of cervical cancers 90% of infections clear in 7,000 cancers o Cancers with known environmental carcinogens tend to have higher mutational burden o Seems especially true for those where chronic exposure (consistently high) over many years (lifestyle?) o Childhood cancers vs those more commonly found in older people  Mutational signatures differ across cancer types o Individual base substitutions analysed in context of neighbours, groups of mutations with similar base substitutions identified (‘signatures’) o 21 signatures described across 30 cancer types. o Some (e.g. #3) represent different base substitutions evenly, whilst some (e.g. #10) are remarkably specific. o Signatures may be associated with age, exposure to particular mutagens, or reflect defects in specific processes e.g. DNA repair/proliferation. o Some signatures are present in many cancers (e.g. #2: APOBEC cytidine deaminases) others specific to tumour types (e.g. #7: melanoma) KEY LEARNING OUTCOMES Cancers arise through cumulative mutations providing selective advantage Driver mutations can increase rate of subsequent mutations (mutator phenotype) Primarily alterations in DNA sequence, but epigenetics can be important too Importance of oncogenes and tumour suppressors Exogenous vs endogenous: although some cancers appear a result of hereditary genetics and/or error probability connected to cell replication (“bad luck”), most require additional exposure to carcinogens (the Cancer Lottery). Particular carcinogens are more likely to cause cancer in certain tissues, related to exposure Different carcinogens can cause DNA damage through particular mechanisms, which can result in characteristic mutational signatures in defined sets of genes. Different tumour types have characteristic levels of mutational burden Mutational ‘signatures’ can be common between individuals with the same cancer or across cancer types LEARNING OUTCOMES Understand and describe the mechanisms of gene regulation throughout transcription and translation Understand how cancer is caused by dysregulated cell growth and/or death Understand and describe how changes in regulation of gene expression can lead to cancer (e.g. altered cell survival, proliferation, invasion and metastatic behaviour) = ALL GENE EXPRESSION The process by which the information stored in our DNA is converted into a functional product (protein) Highly regulated and complex process (~25,000 human genes) o E.g. >200 different cell types present in the body, but all of the cells have the same DNA The expression of specific genes determines the identity of the cell CENTRAL DOGMA OF MOLECULAR BIOLOGY Gene expression is controlled by a variety of mechanisms at multiple levels MECHANISMS OF REGULATION Transcription Post-transcription Translation Post-translation Epigenetic TRANSCRIPTION Portions of DNA sequence are transcribed into RNA o Catalysed by RNA polymerase Signals encoded in DNA tell RNA polymerase Where to start and stop REGULATION OF TRANSCRIPTION INITIATION Cis-regulating elements: DNA in the vicinity of the structural portion of a gene that are required for gene expression Trans-activating factors: Factors that bind to the cis-acting sequences to control gene expression (Transcription factors) Help position RNA polymerase at promoter site and initiate transcription o Positive regulators of gene expression TRANSCRIPTION FACTORS Hundreds of different transcription factors have been discovered; each recognises and binds with a specific nucleotide sequence in the DNA A specific combination of transcription factors is necessary to initiate transcription of a gene. So the presence/availability of the required transcription factors regulate transcription initiation Transcription factors are also themselves regulated by signals produced from other molecules  o e.g. Estrogen receptor (ER) is a transcription factor that is regulated by hormones (estrogen). In other words, hormones are capable of activating certain genes, in this case, ER controls cell division and differentiation in the ovary, breast, and uterus REGULATION BY TRANSCRIPTION FACTORS Spatial regulation: expressed in a tissue - specific manner Temporal regulation: expressed at a specific time in development Activity regulation: o Protein modification (e.g. phosphorylation) o Activated by ligand binding o May be sequestered until an appropriate environmental signal allows it to interact with the nuclear DNA The binding sites for transcription factors are not always close to the promoter RNA polymerase requires the presence of general transcription factors before transcription can begin However… Transcription initiation alone does not account for gene expression i.e. an on/off switch is not enough Transcription elongation is tightly coupled to RNA processing (post- transcriptional regulation) Such mechanisms allow a cell to fine-tune gene expression rapidly in response to environmental changes POST-TRANSCRIPTIONAL REGULATION Further processing of the RNA The transcript is capped and poly -adenylated RNA splicing then removes the non -coding parts of the transcript (introns) so that only the coding sections (exons) remain Alternative splicing o Variations in mRNA (and proteins) o One gene can encode for more than one protein (average human gene is thought to code for 3 proteins) o For example, calcitonin is alternatively spliced in the thyroid and neurons (same gene, different protein) o Genetic diversity (biological complexity and survival) Varying rate of transport of mRNA through the nuclear pores Varying longevity of mRNA o mRNA can last a long time and can continue to produce protein in the absence of DNA o For example, mammalian red blood cells eject their nucleus but continue to synthesise haemoglobin for several months Degradation of mRNA o Ribonucleases destroy mRNA o Hormones stabilise certain mRNA transcripts NON-CODING RNA AND POST-TRANSCRIPTIONAL REGULATION Only a small fraction (3-5%) of RNA code for proteins (mRNA) A significant amount of the genome is transcribed into non-coding RNA (ncRNA). e.g. majority (80%) of RNA is ribosomal RNA (rRNA) MiRNA AND POST-TRANSCRIPTIONAL REGULATION miRNA can bind to protein complexes to form RNA-induced Silencing Complexes (RISC) to inhibit mRNA translation o these small ncRNA are collectively referred to as small interfering RNA (siRNA) o RISC binding interferes with translation, resulting in down —regulation of gene expression miRNA are a class of ~22 bp single-stranded RNA molecules that regulate gene expression post- transcriptionally TRANSLATION The mRNA sequence is decoded in sets of 3 nucleotides (codons) Each codon has a complementary 3 nucleotide binding site (anticodon) on a transfer RNA (tRNA) which carries an amino acid Ribosomes help match the tRNA to the correct codon and catalyses the formation of a polypeptide chain (protein) REGULATION OF GENE EXPRESSION VIA TRANSLATIONAL CONTROL Physical regulation (blockage) o Proteins (even miRNA) that bind to mRNA (via specific sequences) can prevent ribosomes from attaching and thus prevent translation of certain mRNA molecules Initiation factors o These bind to and help assemble the ribosome complex to initate translation o The availability of these factors are also regulated and are produced when certain proteins are needed ▪ E.g. translation initiation factors are simultaneously activated in an egg following fertilization tRNA heterogeneity and codon usage bias o the relative abundance of tRNA can vary between tissues o coupled with preferential use of specific codons, this can regulate the rate if translation POST-TRANSLATIONAL MODIFICATIONS Increases the functional diversity of the proteome The modifications include: o Covalent addition of functional groups or proteins o Proteolytic coverage of regulatory subunits o degradation of entire proteins (proteasomes bind ubiquitinated proteins and degrade them) molecular chaperones help guide the folding of many proteins post-translational modifications can result in protein activation (some proteins are not active when first formed) POST-TRANSLATIONAL REGULATION OF GENE EXPRESSION EXAMPLES OF POST-TRANSLATIONAL MODIFICATIONS Phosphorylation: phosphate group, usually to Ser, Tyr, Thr or His Glycosylation: carbohydrates, usually Asn, hydroxylysine, Ser, or Thr Methylation: methyl group, usually at Lys or Arg residues Acetylation: acetyl group, usually at the N-terminus of the protein Alkylation: alkyl group (e.g. methyl, ethyl) Biotinylation: Acylation of conserved Lys residues with a biotin appendage Glutamylation: Covalent linkage of Glu residues to tubulin and some other proteins Glycylation: Covalent linkage of 1 to > 40 Gly residues to the tubulin C-terminal tail of the amino acid sequence Isoprenylation: isoprenoid group (e.g. farnesol and geranylgeraniol) Lipoylation: lipoate functionality Phosphopantetheinylation: 4'-phosphopantetheinyl moiety Sulfation: sulfate group to a Tyr EPIGENETIC REGULATION OF GENE EXPRESSION Alterations that cause a change in gene expression but doesn’t involve any changes in the DNA sequence DNA methylation o Addition of a methyl group to cytosine at CpG islands (regions of the genome with high frequency of CG sites) o Inhibits transcription by preventing access to promoters o Plays a roll in silencing tissue-specific genes Histone modifications o Acetylation is associated with euchromatin ("open for transcription“ or active) o Deacetylation is associated with heterochromatin ("closed“ or tightly wound DNA) Histone acetyltransferases, histone deacetylases (HDACs) and others regulate these modifications Some cancer cells overexpress or aberrantly recruit HDACs o hypoacetylation, condensed chromatin structure and reduced transcription HDAC inhibitors are valuable cancer therapeutics, they can increase transcription, and lead to cell cycle arrest and apoptosis Epigenetic alterations are heritable traits REGULATION OF GENE EXPRESSION THROUGHOUT TRANSCRIPTION AND TRANSLATION LEARNING OUTCOME 2 - UNDERSTAND HOW CANCER IS CAUSED BY DYSREGULATED CELL GROWTH AND/OR DEATH CANCER Cancer cells reproduce without restraint and colonize foreign tissues Most cancers derive from a single abnormal cell (epigenetic or genetic) However, a single mutation is not enough to cause cancer DNA repair mechanisms and redundancy are able to prevent some of these mutations from causing damage There are multiple stages of development from mildly aberrant cells to cancer MULTIPLE STAGES OF TUMOUR DEVELOPMENT Initiation, promotion, progression, invasion and metastasis  LEARNING OUTCOME 3 - UNDERSTAND AND DESCRIBE HOW CHANGES IN REGULATION OF GENE EXPRESSION CAN LEAD TO CANCER (E.G. ALTERED CELL SURVIVAL, PROLIFERATION, INVASION AND METASTATIC BEHAVIOUR) GENE EXPRESSION AND CANCER Cancer is a disease of dysregulated gene expression that imparts a survival advantage to the cell o i.e. cancer growth depends on defective control of cell death or cell differentiation Alterations can occur at all levels of gene expression o e.g. histone acetylation, activation of transcription factors, increased mRNA stability, increased translational control and protein modification HOW DO CELLS LOSE CONTROL OF CELL GROWTH AND/OR DEATH? GENE EXPRESSION AND CANCER Proto-oncogenes – genes that stimulate cell growth and division in normal cells o these become oncogenes in cancer (gain of function mutation) Tumour-suppressor genes – genes that inhibit cell division in response to DNA damage and allow repair to occur o These usually lose their function in cancer (loss-of-function) Changes in gene expression can have different effects, for example: HOW DO CELLS LOSE CELL POLARITY AND MISCOMMUNICATE? EPITHELIAL-MESENCHYMAL TRANSITION (EMT) The process by which epithelial cells lose their cell polarity and cell-cell adhesion, and gain migratory and invasive properties Occurs naturally (e.g. tissue repair), but also important for initiation of metastasis in cancer WHAT ARE THE PRACTICAL APPLICATIONS OF STUDYING GENE EXPRESSION IN CANCER? GENE EXPRESSION PROFILING IN CANCER Gene expression profiling: capturing total gene activities, both increases and decreases, across a genome as patterns of gene expression Allows us to define different subtypes of cancer based on their gene expression profiles Useful for: o Treatment selection: identify which specific pathways are deregulated in the tumour and treat with therapies that target that pathway. e.g. hormone therapy for ER+ breast cancers o May predict cancer patient survival and prognosis o Increasing understanding the molecular pathways that underlie cancer An example – gene expression profiling and treatment selection in breast cancer Breast cancer is the leading cause of cancer-related mortality in women worldwide Breast cancer is a heterogenous group of cancers with characteristic molecular features, prognosis and responses to available therapy Based on comprehensive gene expression profiling, breast cancer tumours are classified into 3 types: o Luminal ▪ ER and PR positive ▪ Hormonal intervention o HER2+ ▪ ERBB2 over-expression ▪ Anti-HER2 therapy o Basal-like ▪ Triple negative ▪ Most difficult to treat BUT THERE’S MORE TO BE GAINED FROM GENE EXPRESSION PROFILING An example of good genes gone bad Transcriptomic changes can be used to assess cancer progression SO WHY HAVENT WE CURED IT YET? – TUMOUR HETEROGENEITY ADDS COMPLEXITY Inter-tumour heterogeneity: variation between patients. Different morphology types, expression subtypes, or gene expression patterns et. Intra-tumour heterogeneity: variation within a tumour. Different morphology AND different at the molecular level (tumour subpopulations) Cancer cells frequently display startling heterogeneity for various traits related to tumorigenesis: angiogenic, invasive, and metastatic potential o Explanations: clonal and cellular diversity for genetic and epigenetic alterations, adaptive responses or fluctuation in protein levels and activity of signaling pathways AN EXAMPLE: GENE EXPRESSION PROFILING IN MYELOFIBROSIS, A BONE MARROW CANCER Myelofibrosis is a bone marrow cancer where there is extensive scarring in the marrow that ultimately results in an inability to make blood cells It belongs to a group of haematological malignancies called myeloproliferative neoplasms (taught later in the semester by Professor Wendy Erber) Diagnosis and monitoring requires an invasive bone marrow examination Can we develop a simple blood test to assess myelofibrosis? We performed gene expression profiling of platelets from patients using next- generation sequencing o a high throughput technique that allowed us to examine expression of >95% of the human transcriptome BENEFITS OF GENE EXPRESSION PROFILING SUMMARY - Cancer is a complex disease LECTURE OUTLINE Definition and uses of (cancer) epidemiology Descriptive epidemiology o Interpretation of cancer statistics including trends over time o Review of cancer statistics in Australia, compared with the rest of the world Analytical epidemiology o Types of study designs and associated strengths and weaknesses o Measures of association o Causation EPIDEMIOLOGY Epidemiology = the study of the distribution and determinants of health-related states or events (incl. disease), and the application of this study to control of diseases and other health problems Epidemiology is based on two principles o Populations ▪ Who, where, when (person, place, time) ▪ Defined geographically, socially, biologically, time ▪ Predictions at a population level o Comparisons ▪ Differences between groups Disease/no disease; exposed/not exposed Descriptive epidemiology o Examining the distribution of disease in a population, and observing the basic features of its distribution in terms of time, place, and person. o Useful for: ▪ Allocating resources ▪ Planning programs ▪ Hypothesis development Analytic epidemiology o Testing a specific hypothesis about the relationship of a disease to a putative cause, by conducting an epidemiologic study that relates the exposure of interest (determinant) to the disease of interest. o Useful for: ▪ Hypothesis testing ▪ Causal inference TERMINOLOGY Incidence - rate or risk of developing a condition Number of new cases in a certain period of time Cumulative incidence (risk): disease events per person Incidence rate: disease events per person time at risk Useful for understanding risk (cumulative incidence) and speed events occur (incidence rate) Prevalence - proportion of population with a condition o Number of existing cases at a point in time or within a defined period ▪ Point: existing cases at a single point in time ▪ Period: existing cases over a set period o Useful for planning health services Population at risk o Most measures (incidence/prevalence) refer to cases relative to the ‘population at risk’ (PAR) o In mathematical terms; ▪ Cases make up the numerator ▪ PAR is the denominator ▪ We are interested in Cases/PAR ▪ Individuals in the denominator must have the potential to become a case ▪ Eg. Ovarian Ca , prostate Ca , mesothelioma (exposed to asbestos) DESCRIPTIVE EPIDEMIOLOGY Cancer incidence in Australia, understanding age -standardized rates CANCER INCIDENCE 1. NEW CASES  2. CRUDE RATE Number of new cases does not take into account changes in the number of people in Australia over time.  calculate incidence rate Crude incidence - number of cases divided by number in population does not take into account the changes in the age structure of the population (ageing of population) Many diseases (inc cancer) are age dependent. o biology o cumulative exposures to compare rates over time (or between countries) need to control for age 3. AGE-STANDARDISED RATE Calculated by multiplying each age-specific (5 year intervals) incidence rate of a population by a standard population* of the same age groups to yield expected number of cases. These are then added across age groups and the sum is divided by the total standard population. Example: hypothetical country with crude mortality rate of 1,069/100,000 persons (130916/12,340,00). ASR = 582/100,000 CANCER INCIDENCE NUMBERS V ASR Age-standardised incidence rate of all cancers ↑from 383/100,000 (1982) to 486/100,000 (2021). Still other factors that have lead to increase over time CANCER INCIDENCE Common cancers o Different types of cancer are different diseases o Each cancer type has its own descriptive epidemiology ▪ Rates (& trends) of cancer differ by age and sex CANCER INCIDENCE IN AUSTRALIA Most common cancers (incidence) o 1982 – 2020 o All persons o All ages CHANGING PATTERNS OF CANCER IN AUSTRALIA (1982 - 2017) Increase in cancers largely attributed to the rise in the number of prostate and breast cancers. Reasons? o 1) improved diagnoses through screening programs improved technologies ▪ Screening captures all cancers, including indolent or dormant cancers that may not cause harm o 2) Latency from past exposures o 3) Changing exposures SCREENING TESTS Screening tests are performed to detect potential cancers in people who do not have any symptoms of disease. But limited to risk groups Screening tests are not considered diagnostic but are used to identify a subset of the population who should have additional testing to determine the presence or absence of disease. Examples o Mammography: Breast cancer, every 2-yrs for women between 50 & 74 o Faecal immunochemical test: Bowel cancer, every 2-yrs for anyone between 50 & 74 o HPV (Pap) testing: Cervical cancer, every 2 - 5yrs for women 25 - 74 o Prostate specific antigen (PSA): Prostate cancer – men >50yrs o CT: Lung cancer, for high risk groups (smokers) EARLY DETECTION CAN INCREASE CHANCE OF SURVIVAL/REMISSION SCREENING AND CANCER INCIDENCE Figure: trend in breast cancer incidence and the introduction of organized mammographic screening programs CANCER MORTALITY Mortality rates are affected by: o incidence o survival ANALYTICAL EPIDEMIOLOGY Searching for causes Search for a relationship between risk factor(s) and disease o Obtain a valid and precise estimate of effect of an exposure on the occurrence of disease o Determine if the relationship is causal ▪ Many things associated with cancer ▪ Association ≠ causation o Key feature is the inclusion of comparison group(s) ▪ With v without cancer (case-control) ▪ Exposed v unexposed (cohort studies) ECOLOGICAL STUDIES The data on exposure and outcome are aggregated data rather than individual data Aggregated - Summary measure of characteristics of whole populations o e.g. % tobacco sales for each state rather than number of cigarettes purchased by individuals in each state o % lung cancer death for each state Look for correlation between exposure and outcome ECOLOGICAL FALLACY The association at aggregate level may not apply at the individual level Ecological, and other descriptive, studies can be hypothesis generating but are NOT hypothesis testing CASE-CONTROL STUDIES Starting point is the disease of interest Study begins AFTER the outcome has occurred (generally) Cases are chosen because they have the outcome of interest Controls don’t have the outcome of interest (at time of recruitment) o But ARE from population at risk Proportion of exposed subjects are compared in the two groups (odds of exposure – odds ratio) Efficient for rare diseases (eg. cancer) Good for diseases with long latency (eg. cancer) Can study several exposures Breast cancer and shift work study Justification: Some evidence that shiftwork that involves circadian disruption is carcinogenic to humans (IARC 2B: Probably carcinogen) Study design: Population-based case–control study conducted in Western Australia from 2009 to 2011 Subjects: 1205 incident breast cancer cases from WA Cancer Registry and 1789 frequency age-matched controls from the WA electoral roll. Methods: Women were mailed a questionnaire regarding demographic characteristics, reproductive history and lifestyle factors (e.g., alcohol intake, smoking, physical activity and sleep), and completed two tests on circadian rhythm. Participants also provided information on each job they had held for at least 6 months. Groups were compared for markers of shift work and sleep disturbance Results: A small increase in risk was suggested for those ever doing the graveyard shift (work between midnight and 0500 hours) and breast cancer (odds ratio (OR)=1.16, 95% confidence interval (CI)=0.97–1.39). COHORT STUDIES A cohort refers group of individuals defined by an event or common characteristic: o Exposure to a specific agent (eg. Hiroshima, Chernobyl) o Occupation (eg asbestos mining) o Location (eg Framingham, Busselton) o Age cohort (eg Raine pregnancy cohort, 45 -up study) o Other Cohort members are disease free at the start o Compare disease incidence between exposed and un -exposed Participants classified according to exposure status and followed-up over time to ascertain outcome Can be used to find multiple outcomes from a single exposure Appropriate for rare exposures Ensures temporality (exposure occurs before observed outcome) Best way to ascertain both incidence and natural history of a disorder WA AIR POLLUTION AND LUNG CANCER STUDY Cohort study: Health in Men study (HIMS) o 11,679 men >65yo. Recruited 1996-99. Followed until 2018 o Follow-up only included men without lung cancer at recruitment o Lung cancer incidence identified from WA cancer registry o Average annual air PM2.5, NO2 and BC concentrations estimated at each participant’s home address using a land-use regression model o Statistical models ▪ examine how specified factors (air pollution) influence the rate of a particular event happening ▪ controlled for smoking, SES, and other pollutants COHORT VS CASE-CONTROL STUDIES STUDIES Limitations of epidemiological research To study the association between a potential risk factor and a disease one has to aim for: o Accurate diagnosis of disease ▪ Disease (mis)classification o Accurate exposure assessment ▪ Exposure assessment is a perennial problem o Other problems ▪ Bias in the study design ▪ Confounding (known or unknown factors that can distort associations) Associated with both exposure and outcomes Bias Systematic error built into the study design o Any trend in the collection, analysis, interpretation, publication or review of data that can lead to conclusions which are systematically different from the truth. o Selection Bias ▪ Recruitment bias ▪ Response bias ▪ Loss to follow-up o Information Bias ▪ Recall ▪ Social desirability ▪ Interviewer/observer bias ▪ Hawthorne effect Confounding Establishing causation First consider the ABCs o (Accident (chance), Bias and Confounding) Weight of evidence o 1. Strength of association o 2. Consistency o 3. Temporality o 4. Biological gradient (dose response) o 5. Plausibility CLASSIFYING CARCINOGENS International Agency for Research in Cancer (IARC) is interdisciplinary, bringing together skills in epidemiology, laboratory sciences and biostatistics to identify the causes of cancer so that preventive measures may be adopted and the burden of disease and associated suffering reduced. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans o Class 1: Carcinogenic to humans o Class 2a: Probably carcinogenic to humans o Class 2b: Possibly carcinogenic to humans o Class 3: Not classifiable as to its carcinogenicity to humans o Class 4: Probably not carcinogenic to humans IARC Assessment Exposure data Studies of cancer in humans o Sufficient evidence o Limited evidence o Inadequate evidence o Evidence suggesting lack of carcinogenicity Studies of cancer in animals o Sufficient evidence o Limited evidence o Inadequate evidence o Evidence suggesting lack of carcinogenicity Mechanistic data Cancer Epidemiology - in summary - Provides us with information on the distribution of cancer and the risk factors for cancer Uses for practicing doctor Prognosis of diseases Answers to the question of “why me?” Uses for policy makers Planning services Screening programs Preventive strategies Much of what we know about the patterns and causes of cancer comes from epidemiological studies SIGNAL TRANSDUCTION The process by which a cell converts an extracellular signal into a cellular response Involved in: Cell response to environment Cell to cell communication Intracellular homeostasis Leads to alteration in cellular activity Proliferation, apoptosis, metabolism, angiogenesis, migration, invasion, metastasis, etc. ESSENTIAL SIGNAL TRANSDUCTION PATHWAY COMPONENTS Ligands: Growth factors - e.g. Epo, EGF, PDGF, IGF I/II, TGF-α/β Neurotransmitters – e.g. epinephrine, histamine, serotonin Hormones – e.g. oestrogens, androgens, prostaglandins Cytokines – e.g. Interleukins, chemokines Odorants, photons etc Receptors: (i) Cell surface receptors – Cytokine/growth factor receptors,tyrosine kinase receptors, G-protein coupled, ligand- gated ion channels (ii) Intracellular receptor – hormone receptors (e.g. AR, ER, PR) 2nd messengers: cAMP, Ca2+, DAG, PIP3/4, kinases, phosphatases etc. Targets: Transcription factors, translation factors, chaperones, actin/myosin filaments, enzymes etc. SIGNALLING CIRCUITS ARE INTEGRATED WITH CROSS-TALK BUT ALSO HAVE HUBS FOR SPECIFIC FUNCTIONS SIMPLIFIED CELL SIGNALLING PATHWAYS - COMMON PATHWAYS/NETWORKS GROWTH FACTOR / CYTOKINE RECEPTOR SIGNALLING – ACTIVATION/INITIATING THE PROCESS Growth factors and cytokines initiate signalling from receptors by specifically binding to and promoting active receptor configuration – can be by forming complexes or altering the receptor complex orientation. THE EGF-RECEPTOR (HER2) FAMILY – TARGETS FOR CANCER Family members are over-expressed of have activating mutations in breast, gastric and lung cancer. Two prominent pathways activated by the EGF/Her2 family of GF are the ras-raf-Erk and the PI3K/Akt pathways. Inhibitors of the signalling have been developed as either antibodies (e.g. Herceptin/Trastuzumab, used in breast cancer) or tyrosine kinase inhibitors (e.g. Erlotinib, used in NSCLC) Down-Stream Targeting of Commonly Activated Pathways – ras/raf/Erk One common pathway activated by many cell activators that promotes cell proliferation – a hallmark of cancer – is the ras-rafErk pathway THE INCIDENCE OF ACTIVATING MUTATIONS IN THE EGF/ EGFR -RAS-RAF- ERK PATHWAY IN CANCERS Different components of the pathway are mutated in different cancers, i.e. in most lung cancers there are high levels of EGFR (right at the top of the pathway), while in melanoma there is a common activation of raf (specifically B-raf). So targeting down-stream of raf would potentially be widely applicable to many cancers TARGETING THE RAS-RAF-ERK PATHWAY FOR CANCER TREATMENT Several specific drugs have been developed that target the pathway with the most successful being those targeting the MEK1/2 SPECIFIC CASE STUDY OF TARGETING RAS- RAF-ERK PATHWAY IN MELANOMA Resistance to Therapies Targeting the B-RAF in Melanoma Down-Stream Targeting of Commonly Activated Pathways – Viability (PI3K/Akt, apoptosis) AKT IS A CENTRAL PLAYER IN THE VIABILITY PATHWAY OF CANCER CELLS. Another hallmark of cancer is elevated survival signalling, and a central player in this pathway is the S/T kinase Akt. Akt is activated by many different upstream receptor systems and feeds into multiple downstream pathways. Akt also controls protein synthesis which is also a pathway targeted in cancer as the increased growth rate of cancer cells means they have to produce more protein. DOWN-STREAM TARGETING OF COMMONLY ACTIVATED PATHWAYS – VIABILITY (PI3K/AKT, APOPTOSIS) Detail of the immediate Akt pathway and inhibitors that affect its activity TARGETING THE AKT PATHWAY IN CANCERS Inhibitors in red are under development. Inhibitors in blue are in clinical use. THE CASE OF CHRONIC MYELOID LEUKEMIA (CML) AND THE BCR-ABL GENE FUSION BCR-ABL INHIBITORS FOR CML The BCR-Abl fusion protein is a constitutively active kinase. A novel drug STI571 (Glivec/Gleevec/Imatinib mesylate/CGP57148) has been able to interfere with the kinase activity of BCR-Abl. It induces apoptosis in BCR- Abl+ cells by down regulating Bcl-X. The specificity of the drug is highest for the abnormal BCR-Abl kinase, and much less effective against normal kinases in the cell. The drug, therefore, targets the cause of the leukaemia (unlike conventional chemotherapy). Remission rates for CML patients in blast crisis is almost 100% !!! Resistance to Imatinib/STI571 Mostly Due to Mutation of BCR-Abl (T315I) Imatinib fits into the ATP binding pocket of wild-type BCR-Abl but when Threonine 315 is mutated to Isoleucine it can no longer fit, as the Isoleucine protrudes into the space that part of the Imatinib would fit. Mg-ATP can still bind into the pocket so the T315I BCR-Abl is still active. Inhibitors Targeting the Signalling Pathways of the Hallmarks of Cancer CONCLUSIONS Signal transduction is an essential cellular process which transmits messages from the cell surface to the cytoplasm &/ or nucleus of cells Cell signalling regulates many biological processes such as proliferation, apoptosis, angiogenesis, migration Hyperactivation of signalling pathways (by aberrant expression &/or mutation) is a common cause of carcinogenesis Targeting specific signalling components is an effective strategy for the treatment of many cancers SENESCENCE, CELL CYCLE, DNA REPLICATION AND TELOMERES One hallmark of cancer is “immortalization” - endless replication capacity. Most normal cells will divide (replicate, DNA synthesis) in culture for a limited number of times, usually 30-50 division cycles. These cells then enter a phase called senescence where they stop proliferating but they also do not start dying. These cells are characterized by having short telomeres (ends of chromosomes). After an immortalizing event, that results in increased telomere length, the cells will again start dividing. Cancerous cells divide continuously in culture and do not enter a senescence phase as they have high telomerase activity TELOMERE LENGTH AND CELL TYPE Some of your normal cells need to replicate many times: 1) Stem cells need to be able to replicate throughout your life, but at a very low rate, to replace specific cell types. 2) Germ cells need to be able to replicate over generations so we can reproduce, but at a low rate SENESCENCE AND THE CELL CYCLE G0 is the stage of the cell cycle when cells are not dividing. G0 cells can be in senescence or entering terminal differentiation. Mitogens (growth factors) stimulate cells in G0 to enter the cell cycle. The lack of appropriate mitogens/nutrients or specific anti-growth signals will result in a cell exiting the cell cycle. If the cell cycle can not be completed correctly then the cell will enter apoptosis (programmed cell death) and destroy itself. PHASES OF THE CELL CYCLE Inter-phase - no visible characteristics of this stage. Mitotic-phase (M-phase) - can visibly see the cell and chromosomes dividing. G1-phase - Gap phase between end of cytokinesis and beginning of DNA synthesis. Synthesis-phase (S-phase) - DNA is synthesised (replicated). G2-phase - Gap phase between end of DNA synthesis and beginning of mitosis. Mitosis - division/replication of the nucleus. Cytokinesis - division/replication of the cell/cytoplasm. DNA REPLICATION The human genome is 3 X 109 bp long. Placed end-to-end the DNA in each cell is more than 1m long. The average sized cell in the body is 20µm, nucleus is about half that size. To replicate DNA from the start of one chromosome to the end at 50 bases per second would take ~800hr to complete. Replication must start at multiple sites for DNA synthesis to be completed within ~8hr. Only 3 -5 errors are made by the enzymes that replicate DNA during each round of synthesis. – The efficiency rating is therefore 99.999999999%. DNA anti-parallel double helix needs to be unwound – helicase activity. DNA is synthesized only in 5’ to 3’ direction DNA REPLICATION: LEADING AND LAGGING STRANDS THE REPLICATION FORK TELOMERES AND TELOMERASE All cells have a limited ability to replicate themselves. They eventually reach a non-dividing state called “senescence”. At the ends of each chromosome are regions called Telomeres. With each round of cell division, the DNA at each telomere gets progressively shorter. The reduction in telomere length is associated with senescence. An enzyme called telomerase (hTERT) attempts to maintain telomere length. Cancer cells have high telomerase activity enabling cells to keep dividing. DNA replication at the telomeres TELOMERES AND TELOMERASE The average length of the double -stranded TTAGGG repeat varies among species: Mice -------------- 50,000 bp Man -------------- 10,000 bp Telomeres serve at least four functions: 1. Telomeres protect the chromosome ends from degradation and fusion. 2. Telomeres mask chromosome ends from DNA-damage response mechanisms that might trigger apoptosis. 3. Telomeres help position the chromosomes in the nucleus. 4. Telomeres provide a means of maintaining chromosome length through many generations of replication. Telomerase: a ribonucleoprotein with reverse transcriptase activity that catalyzes the extension or lengthening of telomeres. Telomerase caries its own RNA template TELOMERASE IN ACTION Movie showing telomerase recognising “TTTGGG” site (Yeast repeat sequence) with complementary RNA strand and reverse transcribing new DNA to extend the 3’ strand to maintenance of the telomere ends ALT: ALTERNATIVE LENGTHENING OF TELOMERES Unequal Telomeric SCE (Sister Chromatid Exchange). Homologous recombination dependent telomere maintenance, but one daughter cell suffers even shorter telomeres Stand invasion – similar to the D-loop structure at the ends of telomeres (see latter slides) - can lead to initiation of DNA synthesis TELOMERASE AND CANCER TELOMERES ARE NOT FREE AND OPEN STRUCTURE OF THE TELOMERE T-LOOP AND D-LOOP Shelterin complex – Telomere associated protein complexes Protein complexes bind to the telomere and control telomere elongation as well as protect/prevent unwanted responses from ssDNA. TRF1: double-stranded telomeric repeat- binding factor-1 TRF2 (TRF1 and TRF2 bind to the TTAGGG repeat in telomeric dsDNA) RAP1: repressor and activator protein 1 TIN2: TRF interacting nuclear protein 2 (keeps TRF1, TRF2 together – linked to POT1) POT1: protection of telomeres 1 (binds to the ssDNA) TPP1: POT1- and TIN2-interacting protein (keeps TRF1, TRF2 complexes and POT1 together) Shelterin (TRF1/TRF2/RAP1/TIN2/POT1/TPP1) is required to recruit hTERT complex to the telomere TRF1 AND TRF2 COMPLEXES - FUNCTIONS TRF1 complex: Interacts with TPP1 (via TRF1/POT1) to recruit hTERT to the telomere and regulate telomere extension –when tankyrase is active it polyA- ribosylates TRF1 to release it from telomeres and allows more hTERT in to elongate the telomeres. TRF2 complex: Prevents NHEJ and homologous recombination from the ssDNA at the telomere, and interact with TRF1 complexes – prevents DNA damage checkpoint activation PATHOLOGY OF DEFECTIVE TELOMERASE FUNCTION Dyskeratosis congenita (DKC): Produces short telomere. Rare progressive congenital disorder with a highly variable phenotype, sometimes resembling premature aging, initially affecting skin, progressing to bone marrow failure and early mortality. Werner syndrome (WS) / Adult Progeria: Rare, autosomal recessive syndrome, characterized by premature aging. The WRN gene produces a helicase (unwinds DNA) primarily during repair of double strand breaks and during stalled replication Bloom syndrome (BS): Rare autosomal recessive disorder characterized by short stature, genomic instability, and development cancer early in life. The BS gene (called BLM) is a DNA helicase. BS patients exhibit a striking genomic instability including excessive homologous recombination and sister chromatid exchanges (SCE). TELOMERASE KNOCKOUT MICE First generation: “normal” 5th - 6th generation: decreased wound-healing capacity, premature ageing, telomeres very short (even at birth), and have an increased risk of developing cancer. When crossbred with mice with deletion of inhibitors of cell cycle inhibition (MDM2), cancer incidence is reduced: allows shortened telomeres to activate p53 (inhibited by MDM2) more and stop cell cycle/induce apoptosis. When crossbred with p53 negative mice, cancer incidence is increased: telomeres are shortened, so increased ALT – more recombination, more mutations – more cancer TELOMERASE INHIBITORS Competitive RNA binding antisense oligonucleotides: o Antisense oligonucleotides can bind to the RNA component used as a template in the reverse transcriptase (RT) reaction and so prevent telomere extension. Anti-hTERT cancer Vaccines: o Widespread cancer cells hTERT expression makes it an ideal target for anticancer therapies. o High hTERT expression causes its peptides to be presented to T -cells via MHC-I complexes on cancer cells. o hTERT vaccines exploit this to elicit CD8+ and CD4+ T-cell-mediated (via FAS/TRAIL) cancer cell apoptosis. BIBR1532 inhibits RNA template translocation: o Telomere extensions needs multiple RT rounds, and translocation of the template along the newly synthesized DNA; BIBR1532  inhibits this translocation – preventing telomere extension. Nucleoside analogues: o AZT (used in anti-HIV therapy) o Block extension which also blocks translocation/progression along the DNA G-quadruplex-stabilizing ligands: o Act by the same mechanism as BIBR1532 and Nucleoside analogue o Block extension which also blocks translocation/progression along the DNA Conclusions The length of the repeating sequence at the end of each chromosome (telomere) determines how many replication cycles a cell can undergo – mature somatic cells restricted to 30/50 divisions. The biochemical process of DNA replication – specifically the lagging strand synthesis – means that after each round of DNA replication a small section of DNA is not replicated. Telomerase (composed of RNA and proteins) is able to extend the repeating unit of telomeres – and is highly expressed in most cancer cells. Inhibitors of telomerase are being trialed as cancer therapeutics – best responses are seen in combination with other anti-cancer drugs LEARNING OUTCOMES Molecular mechanism(s) of cell cycle regulation and the importance of cell cycle checkpoints. The control of cell cycle during tumourigenesis. The concept of cell cycle dependent chemotherapy for cancer treatment CELL PROLIFERATION = cell division q Regulated / controlled by signals outside (eg. growth factors, nutrient availability) and inside (eg. signalling pathway activity) the cell Cancer: loss or abnormalities of control of cell proliferation This lecture: will focus on G1-S phase transition *Technically: cell growth = cells getting larger, however this term is sometimes used to (inaccurately) describe cell proliferation THE PHASES OF THE CELL CYCLE The mammalian cell cycle can be divided into 4 phases o G1 o S (where DNA is replicated) o G2 o M (mitosis) ▪ prophase, metaphase, anaphase, telophase G0: non-proliferating state of cells. (resting cells, cells that have withdrawn from the cell cycle) CELL CYCLE CHECKPOINTS Cell cycle must proceed in a sequential manner Cell cycle checkpoints are places where the cell cycle can be arrested (surveillance mechanisms) to ensure faithful cell replication THE RESTRICTION POINT During G1, cells are responsive to external (environmental) factors (eg. growth factors, nutrients, other cells) which can either promote progression through G1 or promote cell cycle exit (G0) R point: part of (late) G1 where cells ‘commit’ to cell division. Following R point, cells are not as responsive to environmental factors REGULATION OF CELL CYCLE PROGRESSION Cell cycle progression is regulated by cyclin dependent kinases (CDK ’s) CDK’s are serine/threonine kinases CDK function (and appropriate target recognition) is dependent on cyclins Cell cycle progression occurs via sequential activation / inactivation of CDK/cyclin complexes CDK / CYCLIN COMPLEXES G1 phase: o CDK4/6 : Cyclin D1, D2 or D3 Late G1 (after R point): o CDK2 : Cyclin E1 or E2 S phase entry: o CDK2 : Cyclin A1 or A2 Late S phase: o CDK1 : Cyclin A1 or A2 G2 / onset of mitosis: o CDK1 : Cyclin B1 or B2 CYCLIN ACTIVITY IS MOSTLY REGULATED BY GENE EXPRESSION Cyclin levels (sequentially) fluctuate throughout cell cycle Levels of D-type cyclins are regulated by extracellular signals / activation of signalling pathways CDK4/6 complexes with cyclin D1, D2 or D3 have similar enzymatic activities, however expression of D1, D2 and D3 is differentially regulated REGULATION OF ACTIVITY OF CYCLIN-CDK COMPLEXES Cyclin-CDK complexes are regulated by CDK inhibitors (CDKI’s) Two classes of CDKI’s: o INK4 inhibitors o Cip/Kip inhibitors INK4 INHIBITORS Inhibitors of CDK4 o p16(INK4A) o p15(INK4B) o p18(INK4C) o p19(INK4D) Inhibit activity of CDK4 and CDK6 by: o distorting the cyclin D binding site, reducing affinity for cyclin D binding. o distort ATP binding site, reducing enzymatic activity CIP/KIP INHIBITORS p21(Cip1), p27(Kip1), p57(Kip2) Bind all cyclin-CDK complexes Promote the formation of CDK4/6-cyclin D complexes Function by blocking the ATP-binding site in the catalytic cleft of the CDK (CDK2, CDK1) (cf INK4 inhibitors that block formation of CDK-cyclin D complexes) CELL CYCLE PHASE ASSOCIATED BINDING OF P21/P27 TO CDK COMPLEXES G1 progression: o Early: ▪ low levels of cyclins, high levels of p21/p27 o Early-mid: ▪ Cyclin D levels increase (due to mitogenic stimuli) ▪ p21/p27 bind and promote formation of Cyclin D/CDK4/6 complexes – enhances G1 progression ▪ Cyclin E/CDK2 start to increase but p21/p27 bind and inhibit CDK2 complex activity o Mid-late: ▪ Cyclin E/CDK2 start to increase but p21/p27 bind and inhibit CDK2 complex activity – prevents premature S phase entry o Late G1: ▪ Cyclin E/CDK2 levels continue to increase and there isn ’t enough p21/p27 to inhibit activity ▪ CDK2 becomes activated & phosphorylates p27, targeting it for ubiquitin-mediated degradation ▪ Lack of CDK inhibitors promotes S phase entry RETINOBLASTOMA PROTEIN (RB) IS A NEGATIVE REGULATOR OF G1-S TRANSITION Member of a family of cell cycle regulators – “pocket proteins” RB, p107, p130 Hypophosphorylated in early/middle G1 Becomes progressively phosphorylated by activated CDKs during G1 and throughout the remainder of the cell cycle Hyperphosphorylation of RB inactivates it allowing cell cycle progression Retinoblastoma Protein (RB) inhibits S phase entry G1: RB is hypophosphorylated (active) & is growth inhibitory o Binds and inhibits E2F transcription factors Late G1: Cyclin E/CDK2 activity increases and phosphorylate RB o RB becomes hyperphosphorylated (inactive) o RB releases E2F’s o E2F transcriptionally activate genes required for G1/S phase progression (eg. cyclin E) RB therefore controls the R point * E2F’s are degraded/inactivated as cells enter S phase RB REGULATION OF E2F TRANSCRIPTION Hypophosphorylated RB: o Binds E2F complexes which are bound to the promotors of target genes o Recruits histone deacetylases (HDACs) o HDACs remove acetyl groups from histones o Deacetylated histones are associated with closed chromatin = repressed transcription Hyperphosphorylated RB: o Does NOT bind E2F complexes o Lack of RB enables histone acetylases (HATs) to bind E2F complexes o Histones become acetylated, chromatin opens = activation of target gene transcription COMMITTING TO PROLIFERATION: SUMMARY During G1: o RB is hypophosphorylated (active) and bound to E2F transcription factors. o E2F activity is inhibited. Cyclin D levels are low. o Presence of growth factors will activate growth factor receptors and their downstream signalling pathways, increasing cyclin D levels. o Cyclin D will bind CDK4/6 (with the help of p21 and p27), and active CDK4/6:cyclin D complexes will start phosphorylating RB Later in G1: o RB is increasingly phosphorylated (and inhibited) due to increasing CDK4/6:cyclin D activation. o CDK4/6:cyclin D activity continues to be promoted by p21/p27 taken from unbound p21/p27 stores and stolen from CDK2:cyclin E complexes o Increased numbers of CDK2:cyclin E complexes and the transfer of p21/p27 onto CDK4/6:cyclin D complexes causes increased CDK2:cyclin E activity. This further phosphorylates (and inactivates) RB and also phosphorylates p27, targeting it for degradation. o RB becomes fully hyperphosphorylated completely inhibiting it resulting in release of E2Fs o E2Fs activate transcription of their target genes, including cyclin E S phase entry o Cyclin E levels decline. Cyclin A binds CDK2. E2F’s upregulate expression of genes required for S phase progression. CELL CYCLE PROGRESSION IS REGULATED AT ALL PHASES REGULATION OF CELL CYCLE REGULATORS Some examples… o MYC regulation of cell cycle regulators o Regulation of cyclin D1 levels by signalling pathways o Regulation of p21 by p53 REGULATION OF G1–S CELL CYCLE TRANSITION IS CONTROLLED BY MULTIPLE PROTEINS AND PATHWAYS. REGULATION OF G1–S TRANSITION 1) MYC REGULATION OF CELL CYCLE REGULATORS MYC transcription factor o Family of bHLH transcription factors o Activates transcription in partnership with MAX o MAD/MAX complexes repress MYC target genes MYC/MAX: o Increases expression of cyclin D2 o Increases expression of CDK4 o Increases expression of CUL1 (which degrades p27) o Increases expression of E2F1, E2F2, E2F3 MYC/MIZ1 o Represses expression of p15, p21 and p27 Too much MYC (eg. MYC gene amplification) promotes (uncontrolled) cell cycle progression REGULATION OF G1–S TRANSITION 2) REGULATION OF CYCLIN D1 LEVELS Many cell signalling pathways activate expression of cyclin D1 When you read that “activation of signalling pathways stimulates cell proliferation”, this is one of the mechanisms Some cancers have abnormally increased activity in signalling pathways which drives cancer proliferation (eg. HER2 gene amplification in breast cancer) Signaling pathways that drive cell proliferation: o MAPK, PI3K, JAK/STAT, NF-kB, Hedgehog, WNT and more REGULATION OF G1–S TRANSITION 3) REGULATION OF P21 EXPRESSION BY P53 p53 o Transcription factor o Functions as a homotetramer o Is activated following DNA damage o Upregulates p21Cip1 expression to arrest cell proliferation until DNA damage is repaired, or to induce cell death Loss of p53 is one of the most common events in human cancer Can occur via gene deletion, or by acquisition of inactivating mutations ALTERATIONS IN THE EXPRESSION AND FUNCTION OF CELL CYCLE REGULATORS IN CANCER CONTROL OF PROLIFERATION IN CANCER CELLS Cancer cells proliferate more rapidly than their non-malignant counterparts due to defects in cell cycle control Cancer cells are also less responsive to the external environment (growth factors, other cells) compared to non-malignant cells Some cell cycle regulators are more well known as “oncogenes” and “tumour suppressor genes” because they were originally discovered as mutated/altered in cancer COMMON ONCOGENES & TUMOUR SUPPRESSORS INVOLVED IN CELL CYCLE CONTROL Oncogenes: o Genes/proteins that activate normal cell proliferation, but are mutated/activated in cancer resulting in uncontrolled proliferation o Jammed accelerator o Examples: ▪ MYC ▪ RAS ▪ EGFR/ERBB1 ▪ HER2/ERBB2 Tumour suppressor genes: o Genes/proteins that normally prevent cell proliferation, but are mutated/inactivated in cancer resulting in uncontrolled proliferation o Defective brakes o Examples: ▪ TP53 (encodes p53) ▪ RB ▪ CDKN2A (encodes Ink4a and Arf) ▪ CDKN2B (encodes Ink4b) CELL CYCLE REGULATORS ARE COMMONLY MUTATED IN CANCER CELL CYCLE DEPENDENT CHEMOTHERAPY FOR CANCER TREATMENT TARGETING THE CELL CYCLE FOR CANCER THERAPY Some cancer treatments target rapidly proliferating cells BUT these treatments also affect non-malignant cells eg. cells lining the gut, bone marrow cells, hair follicle cells New molecularly targeted drugs are targeting the cell cycle more precisely Cancer cells with cell cycle mutations (eg. CCND1) are more susceptible Control of Cell Proliferation in Cancer: Summary Abnormal cell proliferation o is a hallmark of cancer o may be due to mutations in cell cycle regulatory proteins o may result from abnormal regulation of cell cycle regulators! ▪ Cell death regulation ▪ Signalling pathway activity ▪ Telomerase activity ▪ DNA repair mechanisms Interactions Between Cell Cycle Regulation and Other Cell Processes Cell cycle progression / inhibition is also regulated by o Control of cell death o DNA repair o Activation / inhibition of signalling pathways o Telomerase activity o Function of tumour suppressor genes / oncogenes LEARNING OUTCOMES Control of cell death and different types of cell death Laboratory techniques used to detect apoptosis Discuss mechanisms and signaling pathways involved in apoptosis and their role in cancer WHY CELLS NEED TO DIE Organogenesis and development of complex multicellular tissues Cellular homeostasis and cell damage Response to infectious agents TYPES OF CELL DEATH Programmed cell death – a physiological process where cells are eliminated during development and other normal biological processes o Apoptosis (type I cell death) o Autophagy (type II cell death) o Anoikis – delayed cell death associated with build up of autophagy vesicles o Cornification – epithelial cell specific process to produce outer (dead) layer of the skin o Pyroptosis, pyronecrosis – infection induced death of macrophages o Necroptosis – ‘regulated’ necrosis Necrosis (“accidental” cell death) – a pathological process after exposure to a serious physical or chemical insult APOPTOSIS Morphological features o No loss of membrane integrity o Aggregation of chromatin at the nuclear membrane o Shrinking of the cytoplasm and condensation of nucleus o Fragmentation of cell into apoptotic bodies o Leaky mitochondria due to pore formation Biological features o Strictly regulated process o Energy (ATP) dependent o Ladder pattern of DNA fragmentation (non random) o Prelytic DNA fragmentation o Alteration in membrane asymmetry  Physiological significance o Evoked by physiological stimuli (growth factors etc) o Affects individual cells o Phagocytosis by macrophages or adjacent cells o No inflammatory response NECROSIS Morphological features o Loss of membrane integrity o Swelling of cytoplasm and mitochondria o Total cell lysis o No vesicle formation o Disintegration (swelling) of organelles Biological features o Loss of regulation of ion homeostasis o No energy requirement o Smear pattern of DNA (random digestion) o Postlytic DNA fragmentation Physiological significance o Evoked by non-physiological disturbance o Affects groups of cells o Phagocytosis by macrophages o Significant inflammatory response PATHWAYS TO APOPTOSIS Caspases mediate the events associated with apoptosis They are a family of cysteine proteases PATHWAYS TO APOPTOSIS: EXTRINSIC PATHWAYS TO APOPTOSIS: INTRINSIC REGULATION OF APOPTOSIS Positive modulators (pro-apoptosis) Negative modulators (anti-apoptosis) CRITICAL REGULATION OF APOPTOSIS: BCL-2 FAMILY Inhibitors of apoptosis (anti-apoptosis) o Bcl-2, Bcl-xL, Bcl-w, Mcl1 BH3 only (pro-apoptosis) o Bid, Bim, Bad, Bik, Noxa, Puma Effectors (pro-apoptosis) o Bax, Bak, Bok MOLECULAR STRUCTURE OF BCL -2 FAMILY Anti-apoptotic family members contain four BCL -2 homology domains (BH) as well as a putative trans-membrane domain (TM) responsible for their preferred localization at inner membranes Effectors Bax subfamily resemble BCL-2 closely in structure possessing 3 out of 4 (multiple) BH domains Pro-apoptotic family members “BH3-ONLY” proteins only share one BH3 domain with all other BCL-2 family members BCL2 CONTROL OF APOPTOSIS Positive modulators of apoptosis (inducers) Cytochrome-c o Activates APAF1 Apoptosis protease activating factor 1 (APAF1) o Critical component of apoptosome, cleaves caspase 9 Caspases Apoptosis inducing factor (AIF) o Induces chromatin condensation and DNA degradation Endonuclease G (EndoG) o Facilitates chromatin condensation with AIF Granzyme A (GrA) o Serine protease released by cytotoxic T cells Tumour Suppressor genes o RB1 ▪ RB1 binds to and inhibits E2F family of transcription factors ▪ E2F transcriptionally activates many pro-apoptotic genes eg. BAX, BAD, APAF1, Caspase 9, Caspase 3 o p53 ▪ Transcriptional, and post-translational control of apoptosis. ▪ Enhances expression of pro -apoptotic genes eg. BAX, BAK, PUMA, NOXA ▪ Decreases expression of pro-survival genes, eg. BCL2 Therefore, loss of these tumour suppressors in cancer enables cancer cells to avoid apoptosis NEGATIVE MODULATORS OF APOPTOSIS (INHIBITORS) 1. Bcl -2 family genes: Bcl2, Bcl - XL,, Bcl -w, Mcl - 1 2. Inhibitor of apoptosis proteins (IAPs) - block caspase activation 3. Pro -thymosin - α (ProT α) - blocks apoptosome formation 4. E1B - acts like Bcl2 to bind Bcl2 family effectors Signaling pathways: lots of crosstalk! o PI3K-AKT o NF-kB ▪ Activation leads to enhanced survival ▪ Inhibition of NF-kB promotes apoptosis o ERK-P90RSK o PKC and c-Src, increase survival PI3K/AKT SIGNALING PREVENTS APOPTOSIS To die or not to die? Integrated balance between positive survival factors and negative death signals decide cell fate METHODS FOR STUDYING APOPTOSIS Apoptosis methods are utilized in studies of immunology, embryology, aging, AIDS, neurology and cancer. Protease activity o Caspase 3 activity Membrane alterations – phosphatidylserine translocation o Annexin V: a phospholipid-binding protein with a high affinity for phosphatidylserine DNA fragmentation assay - apoptotic DNA Ladder o Gel electrophoresis DNA strand breaks o TUNEL assay (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) METHODS TO DETECT CASPASE 3 ACTIVITY Caspase 3/7 cut proteins after a DEVD sequence (Asp-Glu-Val-Asp) PHOSPHATIDYLSERINE EXPOSURE MARKS APOPTOTIC CELLS Annexin V (or Annexin A5) is a protein that binds to phosphatidylserine with high affinity and can be labeled with fluorophore to assess apoptosis using flow cytometry DNA FRAGMENTATION ASSAY CAN BE USED TO DETECT APOPTOSIS USING AGAROSE GEL ELECTROPHORESIS A = control with intact genomic DNA B = DNA from cells with extensive apoptosis C = DNA from cells undergoing necrosis TUNEL assay (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) HOW CANCER CELLS AVOID APOPTOSIS Internal pathways o p53 loss of function o Rb inactivating mutations o Myc gene amplification o Bcl2 activation o Bax inactivating mutations o Caspase inactivating mutations The list is long! External pathways/signals o Loss of pro-apoptotic signaling molecules ▪ Death receptor/ligand systems disrupted o Viral infection can prevent apoptosis ▪ HPV (cervical cancer) – E6 protein blocks p53 function ▪ Epstein-Barr virus (EBV) (lymphoma) – mimics BCL2 o Interaction with other cells ▪ Hide from cytotoxic T cells – downregulation of MHC, increase immune suppressive proteins (eg. PD-L1) o Interaction with chemicals APOPTOSIS AND CANCER THERAPY: PRINCIPLES Inducing apoptosis of cancer cells is an ideal therapeutic approach: o Prevents inflammation and damage due to necrotic cell death o Harnesses the cells own apoptotic machinery o But… o Cancerous cells are resistant to apoptosis (hallmark of all cancers!) Need to know affected pathways to produce targeted therapies BCL2 inhibitors as anti-cancer drugs SUMMARY Apoptosis = Programmed Cell Death Intrinsic and extrinsic pathways Tightly regulated and closely linked to proliferation and senescence pathways Cancer commonly linked to changes in apoptotic pathways in cells Reactivating apoptosis pathways is a viable target for cancer therapeutics LEARNING OUTCOMES Understand and be able to correctly apply and discuss the terms; neoplasm, benign, malignant, invasion, metastasis Understand basic pathogenesis and important sequelae of invasion Understand basic pathogenesis and important sequelae of metastasis BACKGROUND Neoplasia: Clonal proliferation of cells which grows in an excessive and uncoordinated way, in comparison to normal tissue, and persists in the absence of the stimulus that first evoked the change o Benign o Malignant BENIGN Benign: Neoplasm lacking the ability to invade and metastasize Macroscopic: generally rounded, well circumscribed, encapsulated tumours Microscopic: banal features (uniform cells, well differentiated, low mitotic rate) Generally innocuous clinical course, amenable to resection; can cause problems depending on site MALIGNANT Malignant: Neoplasm with capacity for local invasion and metastasis Macroscopic: generally infiltrative (crab like), not encapsulated, hard, fibrous Microscopic: pleomorphism, anaplasia, hyperchromasia, mitoses (numerous and abnormal forms) Aggressive clinical course - local and distant effects o high mortality Two important hallmarks of cancer are the capacity for invasion and metastasis Invasion and metastasis are responsible for some morbidity and most mortality due to cancer Invasion and metastasis are important prognostic factors The process is extremely complicated Understanding the complex processes of invasion and/or metastasis is fundamental in the efforts to ‘cure’ cancer Involved molecules are being targeted as novel “anti-cancer drugs” SUMMARY Background/Nomenclature Invasion o Altered cell-cell adhesion o Matrix dissolution o Altered cell-matrix adhesion o Migration/locomotion Metastasis o Intravasation o Vascular dissemination o Extravasation o Colonisation Invasion: the process whereby tumour cells disobey organ boundaries and cross into foreign tissue Metastasis: a secondary tumour established at a site distant from the primary tumour INVASION Invasion is a multistep process o Altered cell-cell interactions o Matrix dissolution o Altered cell-ECM interaction o Mitigration/locomotion STEP 1: ALTERED CELL-CELL INTERACTIONS Normal: tight intercellular adhesions (e.g. E-cadherin homodimers) Tight adhesions lead to transduction of signals o maintain terminal differentiation o regulate growth o prevent cytoskeletal remodelling “contact inhibition” Cancer: tumour cells down regulate adhesion molecules and dissociate from one -another o Loss of “contact inhibition” o De-differentiation o Up-regulated growth o Increased cytoskeletal remodelling E-cadherin is a frequently disrupted molecule in some carcinomas STEP 2: MATRIX DISSOLUTION Normal: tissue compartments are separated by the “extracellular matrix unit (ECM)” ECM consists of basement membrane (BM) and interstitial matrix o BM is a dense matrix of collagens, glycoproteins (e.g. laminins) and proteoglycans o Interstitial matrix is loose matrix of fibrous structural proteins (e.g. collagens, elastins), adhesive glycoproteins and proteoglycans and water These structural elements must be degraded for invasion to occur  Tumour cells either secrete or induce ECM cells (e.g. fibroblasts/inflammatory cells) to secrete proteases Many different classes of proteases are involved o Matrix metalloproteinases (MMPs) o Heparanases o Serine proteases MMPs have a particularly important role MMPs are a family of zinc-dependent protease molecules capable of degrading all ECM components >21 structurally related MMPs Divided based on substrate specificity o Collagenases: degrade collagens type I, II & III o Gelatinases: degrade collagen IV o Stromelysins: degrade collagen IV and proteoglycans o Others… Physiological function o Embryogenesis and growth, uterine cycling and postpartum involution and tissue repair… MMPs breakdown ECM components for physical passage of tumour cells Elaborate growth factors from the ECM o e.g. cleavage products of collagen and proteoglycans, sequestered growth factors Strong correlation between MMP expression and invasive and metastatic potential MMPs are often expressed in cancers (e.g. SCC, breast) but not in their normal or benign counterparts MMPs activity is regulated by tissue inhibitors of MMPs (TIMPs) 4 structurally related proteins (TIMP1 – TIMP4) Block MMP activity by binding to their conserved zinc-binding site Synthesized and secreted by ECM cells as a host response to limit tumour invasion (and also by some cancer cells) Overexpression of TIMPs is correlated with reduced invasive capacity of tumour cells Complex interplay between MMPs and TIMPs determines the degree of ECM degradation STEP 3: ALTERED CELL-MATRIX INTERACTIONS Normal: cells have receptors (e.g. integrins) for ECM constituents (e.g. laminin) along their basal surface Adhesions lead to transduction of inhibitory signals “contact inhibition” o maintain terminal differentiation o regulate growth o prevent cytoskeletal remodelling Integrins are a large family of transmembrane glycoprotein heterodimers with an α and β subunit attached to the intracellular cytoskeleton 24 distinct integrin heterodimers have been described, consisting of 18α and 8β subunits The binding target and the intracellular signal upon binding is determined by the particular combination of the α and β chains Physical effects: regulate the shape, orientation and movement of cells Intracellular signalling cascades which alter gene expression Cancer: Altered integrin expression o Some integrins e.g. αVβ3, almost universally promotes tumour invasion and metastasis o Others e.g. α