Cancer Pathology Notes PDF

PATH3309 – CANCER PATHOLOGY MODULE 1 – GENERAL CANCER PATHOLOGY  A neoplasm, or a tumour, is defined as an abnormal mass of tissue with excessive, uncoordinated and persistent growth despite a lack of any growth stimulus o This definition of a neoplasm excludes physiological...

PATH3309 – CANCER PATHOLOGY MODULE 1 – GENERAL CANCER PATHOLOGY  A neoplasm, or a tumour, is defined as an abnormal mass of tissue with excessive, uncoordinated and persistent growth despite a lack of any growth stimulus o This definition of a neoplasm excludes physiological, reversible tissue growth such as hyperplasia, hypertrophy and inflammation  All tumours have to basic components – clonal cells that originate from a single progenitor cell (can range from a mature cell to a primitive cell), and reactive stroma, or supportive tissue, such as blood vessels and connective tissue that support the abnormal growth of the tumour  Neoplastic growths can be classified and named based on several features such the histogenesis of the growth (morphological and molecular features), the site of origin (e.g. lung, colon, skin), the behavioural features (benign, malignant) or the aetiology (cause) of the tumour  Classification of a tumour based on behaviour depends on the rate of growth and the invasiveness of the tumour: o Benign tumours are relatively slow in their growth and remain localised, meaning they do not spread to distal tissues (e.g. adenomas) o Malignant tumours (cancer) are fast growing and are able to spread to or invade adjacent or distal tissues o Intermediate/borderline tumours are, as the name suggests, more cancerous and abnormal than low grade benign tumours but less so than malignant tumours  Tumours classified by histogenesis are classified based on their morphological and molecular features, and this is often dependent on the type of cell involved – e.g. epithelium, mesenchyme, blood, special cells etc.  There are general rules involved in naming tumours: o Benign tumours have the name of the tissue of origin, followed by the suffix ‘-oma’ – e.g. lipoma, chondroma, adenoma, osteoma o Malignant carcinomas arise from epithelial cells and are named after the cell or tissue type, followed by ‘carcinoma’ – e.g. adenocarcinoma, squamous cell carcinoma o Malignant sarcomas arise from mesenchymal tissues and are named by including the cell or tissue type, followed by ‘sarcoma’ – e.g. chondrosarcoma, liposarcoma  Neoplasms can present as either a primary or a secondary tumour – a primary tumour is the ‘original’ tumour, and can metastasise to a distant site to form a secondary tumour o Every secondary neoplasm has a primary tumour or a site of origin that can be detected or recognised from previous diagnoses, morphology or immunohistochemical profile  The differentiation of a neoplasm is defined as the extent to which the neoplastic cells resemble the corresponding normal parenchymal cells in both their function and morphology o A highly differentiated neoplasm more closely resembles the normal parenchyma than a poorly differentiated neoplasm  Anaplasia refers to the lack of differentiation in a neoplasm and usually produces distinct morphological changes that can be used to ‘grade’ a neoplasm based on the degree of differentiation o Pleomorphism refers to the variation (pleo-) in size and shape (-morphism) o Mitoses are generally abnormal, such as multipolar mitotic spindles which reflects the atypical proliferation of anaplastic cells o Loss of polarity of the cell refers to the disorganised growth of the cell o Abnormal nuclear morphology occurs due to increased nuclear activity and results in an abnormally large and irregular nucleus, as well as a large nucleolus  One grading system that grades tumours and carcinomas based on differentiation is the Gleason grading system; a lower grade carcinoma would be more well differentiated than a higher grade  Metaplasia is defined as the conversion of one cell type to another, and is usually associated with tissue damage and repair – an example of metaplasia is Barrett’s oesophagus, whereby the squamous cells that line the oesophagus are replaced by columnar cells (increase risk of oesophageal carcinoma)  Dysplasia is defined as disordered cell growth that occurs in metaplastic epithelium whereby the structural architecture of the epithelium becomes disordered – does not necessarily indicate cancer but is often found adjacent to invasive carcinomas o A high-grade dysplasia is often called ‘carcinoma in situ’ and is a precursor to an invasive cancer; once a tumour growth breaches the basement membrane it is said to be invasive (cancerous)  There are several ‘hallmarks’ of cancer that allow cancerous cells to persist and continue growing: o Sustained proliferative signalling o Replicative immortality o Evading growth suppression mechanisms that act to prevent uncontrolled proliferation o Evading apoptosis or resisting natural mechanisms of cell death o Invasion or metastasis of the tumour to adjacent or distal tissues o Induction of angiogenesis (growth of new blood vessels)  Other ‘emerging’ hallmarks include immune evasion and deregulation of cellular energetics (different metabolic pathways to produce energy)  Some enabling characteristics or factors that can contribute to development of cancers include inflammation (e.g. H. pylori infection and marginal zone lymphoma), as well as genomic instability (e.g. defective DNA repair mechanisms  One of the major impediments on treatment of cancer is the time taken to detect clinical signs of the cancer – detection of cancers usually occurs after the neoplasm is quite developed o Early detection of cancers or precancerous legions can support treatment – e.g. regular mammograms can detect breasts tumours before lymphatic metastasis  Metastasis is the characteristic feature of cancers and is defined as the formation of a secondary neoplasm at a different site to the primary tumour – in order for metastasis to occur there are several requirements: o The neoplasm needs to penetrate the basement membrane or capsule, move through the extracellular matrix and penetrate vascular channels, survive in these channels, exit into new tissue sites, evoke angiogenesis and form a new neoplastic mass o Some tissues (e.g. brain, liver, lymph nodes) are common sites of metastasis – the site of metastasis can also depend on the site of the primary tumour (e.g. prostate cancers often metastasise into bone)  Carcinomas can spread and metastasise by several means – the ability of neoplasms to move across extracellular matrices allows direct invasion of adjacent tissues, spread across body cavities and intraepithelial spread o They can also spread via haematogenous dissemination (invasion via blood vessels) or lymphatic dissemination (invasion via lymphatic vessels)  Metastasis is responsible for mortality and morbidity associated with cancer – harmful growths in vital organs can lead to death (e.g. liver, lung, brain) or significant morbidity (e.g. bone)  Tumours are often graded as an indication of how ‘abnormal’ the tumour cells appear under a microscope; grading systems can differ based on the type of cancer and, if a grading system is not specified, the tumour is graded between G1 (well differentiated) to G4 (undifferentiated) o The Nottingham grading system is used in breast cancers and grades the tumours based on tubule formation, nuclear grade and mitotic rate; each factor is given a score between 1 and 3, and the sum of the three categories yields a score of 3 – 9 which indicated the level of differentiation of the tumour o The Gleason scoring system is often used in prostate cancer and is based on a primary and secondary pattern of tissue organisation; each pattern is given a score of 1 – 5 based on resemblance to normal tissue, the two scores are added to give a Gleason score between 2 and 10  Cancer staging is an assessment of the likely prognosis of the cancer and is used to direct treatment  o The three components of cancer staging are tumour (size, extent of spread), nodal status (number of lymph nodes, sizes etc.) and metastasis – TNM  The growth fraction refers to the percentage of tumour cells that are actively proliferation as opposed to those that are in the resting phase (G0 phase) of the cell cycle – anticancer agents are most effective on these proliferating cells o Aggressive tumours that have a large growth fraction (e.g. lymphoma, leukaemia) are susceptible to chemotherapy and can even be cured o In the case of tumours with a low growth fraction, a common practice is debulking the tumour by surgery or radiation in order to shift the tumour cells from the G0 phase into the cell cycle, thus increasing susceptibility of the tumour cells to anticancer drug therapy  Different cancers have different mortality rates – the most common cancers are prostate (males) and breast (females) but they both form small percentages of cancer mortality o Similarly, lung cancers and pancreatic cancers have relatively low incidence rates but high mortality rates due to the importance of the lungs and the aggressive nature of pancreatic cancer  Public health is a field of practice that is concerned with prevention of disease in human populations  Epidemiology is a branch of public health that deals with the study and application of the distribution and the determinants of a health-related state or disease in order to control these conditions o It is concerned with entire populations (both healthy and not) and understanding the risk factors that can lead to disease and the measures that can be taken to reduce these factors  There are two main subsets of epidemiology – descriptive and analytical epidemiology o Descriptive epidemiology involves examination of the disease burden, which includes observation of the basic features of its distribution such as time, location and individuals – this is useful for appropriate resource allocation and development of hypothesis o Analytical epidemiology involves testing a specific hypothesis regarding the relationship of a disease and a causative factor by conducting epidemiological studies that relative the exposure of interest to the associated disease  There are numerous terminologies that are important in epidemiology: o The incidence of a disease is defined as the number of new cases in a certain period of time, and is useful for understanding the risk of developing a condition  o The prevalence of a condition is the total number of cases of a condition at a particular point or period of time o Both the prevalence and incidence of a condition are measured relative to the population at risk – meaning that the population considered must have the potential to develop the condition of interest  Descriptive epidemiology of cancer in Australia: o The number of new cases of cancer has increased steadily from ~ 5 000 in 1982 to over 11 000 in 2008 – however, this figure does not incorporate changes in populations o The crude incidence rate (cases per 100 000) has also increased from ~ 300 in 1982 to around 500 cases in 2008 – the crude incidence rate does not consider changes to the demographic of the area (e.g. age)  The age-standardised rate is an important measure in age-related conditions such as cancer – it is calculated by calculating the incidence rate of each age interval per 100,000, and multiplying that by a standard population of the same age group o The slight increase in cancer incidence rates shown in the figure can be attributed to increases in prostate and breast cancer diagnoses o Factors that can contribute to increase incidence rates include improved diagnosis through national screening programs, latencies from past exposures (e.g. mesothelioma) and changing environmental exposures (e.g. UV, radiation)  Different types of cancer are more common in different age and gender demographics than others – e.g. prostate cancers are the most common in males and breast cancers are the most common in females  In contrast to increased incidence rates in Australia, mortality rates have decreased since 1982, and five-year survival rates have also increased o Relative to the rest of the world, Australia has among the lowest mortality-incidence ratios  The lifetime risk of developing cancer is essentially the chance that a person has, given the current incidence and mortality rates, of being diagnosed or dying as a result of cancer  The global cancer rates vary in terms of which cancers are more common than others, as well as the mortality rates of these cancers o These differences in cancer rates can be influenced by genetic predispositions, exposure to risk factors, health care systems in terms or prevention, diagnosis and treatment of cancers etc.  Descriptive epidemiology, such as the statistics shown above, can then be used in order to formulate hypotheses and ask questions regarding risk factors and causes of increased cancer rates etc.  o These questions and hypotheses are then investigated by analytical epidemiological studies  Thus, the key focus of analytical epidemiology is the search for a relationship between a risk factor and a disease (differentiate association from causation)  There are various kinds of study designs used to test these relationships, and they differ in terms of the quality of evidence and level of bias  A ‘study design pyramid’ is shown and reflects the relative ranking of each study design – randomised controlled studies have a relatively high quality of evidence and low risk of bias if performed well  Two study designs that are often used in analytical epidemiology are case-control studies and cohort studies  Case control studies start with a disease of interest and divide a source population into individuals with the disease, and a control population who do not have the disease but are at risk of developing it o Upon assessing prior exposure to the agent of interest, and finding the proportion of exposed subjects in both the control and the disease groups, an odds ratio can be measured, which measures the association between a disease and exposure of interest o This study design is more suitable for rare diseases and those with a degree of latency  Cohort studies start with a study population, and this population is classified according to exposure and then followed-up over time to observe development of the disease of interest and assess relative risk of developing a disease given a certain exposure o This study design is more suited to starting points of rare exposures (e.g. asbestos mining, Hiroshima etc.), and also ensures temporality (exposure before disease)  The two main measures of association between an exposure and resulting disease are: o Relative risk of developing disease based on cumulative incidence and comparison of risk between an exposed cohort and a non-exposed cohort o Odds ratio is a more indirect relative risk measure as it compares the odds of developing the disease having been exposed to an agent compared to not developing the disease having been exposed to that same agent  In order to effectively analyse the association between a risk factor and a disease, there must be accurate diagnosis of the disease, as well as accurate exposure assessment to the risk factor o Both of these can prove to be a limitation in analytical epidemiology of cancer, as there maybe misdiagnoses or inaccurate risk assessments that can distort the studies o Some study designs may also have some degree of bias or other factors that can disrupt the accuracy or reliability of the results   Bias is defined as any systematic error in any stage of the study design that can lead to conclusions different to the true association o Bias comes in many types, such as selection bias (bias in terms of who is included in a particular study) or information bias (what people can recall) etc.  Confounding factors are those that can distort associations between one risk factor and a disease o A prime example of a confounding factor is smoking when trying to assess the association between silica dust (exposure) and lung cancer (outcome)  Establishing causation is done by assessing the weight of evidence that supports the association between a risk factor and a disease outcome – five factors that contribute to the weight of evidence are: o The strength of the association according to the study design o The consistency of the results o Temporality – the exposure must occur before disease development o A biological gradient needs to be established – meaning that there is an association between the dose of the causative agent and the disease o Biological plausibility needs to be established – in other words, is the association scientifically possible  The International Agency for Research in Cancer (IARC) is an interdisciplinary organisation that adopts numerous fields to contribute to identifying common causes of cancer in order to adopt preventative measures o The IARC Evaluation of Carcinogenic Risks to Humans ranks agents from Class 1 (carcinogenic to humans) to Class 4 (probably not carcinogenic to humans) based on the weight of the association between the risk factor and the outcome MODULE 2 – MECHANISMS AND AETIOLOGY OF CANCER  Carcinogenesis is essentially a disease of tissue growth dysregulation, and is the result of a stepwise process whereby normal cells become cancerous  The somatic mutation theory is the most widely accepted theory describing the aetiology of carcinogenesis – it states that cancer is a disease of cell proliferation caused by an accumulation of mutations that arise from a single cell o In other words, a single cell accumulated numerous mutations in genes that control cellular proliferation and the cell cycle, resulting in dysfunctional regulation of tissue growth and uncontrolled proliferation o While it seems that it is extremely unlikely for a cell to develop numerous mutations that result in cancer, a phenomenon that explains how this happens is the mutator phenotype, which states that one mutation increases the risk of more mutations  occurring, and this can be done be increased cell division (more DNA replication), abnormal DNA replication and compromised DNA repair systems  So, after the initial mutation, there is an increased mutation frequency – these mutations can either be lethal to the cell, consequential with no selective advantage (passenger mutation) or provide a selective advantage for the cell (driver mutation) o Selection in terms of cell growth means that the cell is more likely to thrive in its environment than other cells (e.g. evading immune system, bypassing tumour suppressors, angiogenesis etc.) o One study (Wood LD, Parsons DW; 2007) found that the average colon cancer contains 15 driver mutations and 60 passenger mutations  Tumours are often very heterogenous, meaning that the tumour cells may not all have the same genotype – when a cell acquires a mutator phenotype, it is more likely to develop mutations at random, resulting in random pathways being affected o As the cell grows and divides, each daughter cell also has this mutator phenotype and can accumulate its own mutations and so on; the result is a tumour with clonal variants of heterogenous tumour cells  Cellular mutations often occur in one of two ways: o Large scale mutations can involve chromosomal abnormalities, defects in mitosis or meiosis or insertions, deletions or translocations of large fragments of DNA – e.g. individuals with trisomy 21 (Down syndrome) are prone to childhood cancers; translocation 9;22 is seen in 85% of patients with chronic myeloid leukaemia o For the most part, carcinogenic mutations occur on a small scale such as point mutations, where a protein can still be made but their function may be slightly altered; point mutations can lead to frame shifts, which can lead to an abnormal protein or no protein production  Proto-oncogenes are genes that encode proteins involved in stimulation of cell division, blocking differentiation, inhibition of apoptosis etc. – when proto-oncogenes are expressed at higher levels to the point where they may contribute to carcinogenesis, they are referred to as oncogenes o Oncogenes can be the result of amplification of a proto-oncogene, translocation of the gene to a more active promoter region or a mutation resulting in a fusion protein with higher oncogene activity o MYC is an DNA-binding onco-protein that is seen to be overexpressed in >50% of human cancers – its roles involve inhibition of apoptosis and blocking cellular senescence (arrest of the cell cycle); tumours often depend on high MYC expression but also need other mutations to become cancerous  o RAS is a GTPase signalling onco-protein that is activated by proteins called GAPs that ‘switch’ the GTP on or off – a mutated RAS oncogene becomes ubiquitously activated so that signals of cell survival, growth and cell cycle progression are constantly activated; there are three kinds of RAS proteins (KRAS, NRAS, HRAS) but most RAS mutants are KRAS  Tumour suppressor genes are essentially the opposite of oncogenes in that they inhibit carcinogenesis and thus need to be inactivated so that the cell can become cancerous – they often have suppressive or regulatory activity such as controlling proliferation, initiating apoptosis and regulating adhesion o While oncogenes often need a mutation at a single locus (one-hit) for a gain-of- function, tumour suppressor genes often need mutations at both loci (two-hit) for loss- of-function o p53 is an important tumour suppressor gene that is induced by oxidative stress, hypoxia, DNA damage and other factors, and results in induction of apoptosis, DNA repair, induction of senescence and other processes that result in tumour suppression  Epigenetic modifications are those that can alter gene expression and protein translation without changes in DNA sequence – these mechanisms include methylation, histone acetylation, ubiquitination, phosphorylation etc. o These processes can change the structure and appearance of DNA in order to alter the accessibility of DNA to transcription machinery, affecting the enzymes involved and organisation of chromatin o >50% of human cancers have mutations that affect enzymes involved in chromatin organisation  Around 10% of cancers have a hereditary component in their aetiology, but random spontaneous mutations are important in the development of cancer – the risk of developing mutations increase with age o For most cancers, driver mutations are required, some of which are environmental, some hereditary and some replicative  One study analysed over 5 million mutations from over 7 000 cancers in order to identify the mutational burden (prevalence of somatic mutations) of different cancers o Cancers that have known environmental carcinogens (e.g. smoking, alcohol) tend to have higher mutational burdens – esp. those with chronic exposure o Cancers that develop in older people also tend to have higher mutational burdens compared to childhood cancers which have lower burdens – this is because older people tend to accumulate more mutations  The same study attempted to identify characteristic ‘signatures’ in terms of the mutations at the level of base substitutions – over 21 signatures of different proportions of base substitutions were identified across 30 cancer types (e.g. C > T) o Different cancers that have a probable association (e.g. UV radiation, age etc.) were found to have distinct mutational signatures o The mutational signatures are often associated with the probable cause of the cancer such as age, hereditary factors or carcinogenic factors  Carcinogens are agents that increase the rate of mutation and thus increase the risk of developing cancers: o Chemical agents include tobacco smoke, alcohol, asbestos etc. o Biological agents include certain viral, fungal or bacterial species o Radiological agents include radiation or radioactive compounds (e.g. UV, Po-210)  Different carciongens are likely to cause different kinds of cancer depending on the site of exposure and the duration of exposure (e.g. chronic or acute exposure)  Alcohol, for example, increases the risk of cancers of the mouth, pharynx, larynx, oesophagus, bowel, liver, breast and probably stomach as well o The mechanisms by which alcohol (ethanol) can cause cancer include metabolism to acetaldehyde which causes direct tissue damage, direct tissue damage by ethanol, acting as a solvent for other carcinogens (e.g. tobacco) and changing hormone levels such as insulin and oestrogen (associated with breast cancer)  UV radiation is the most common cause of skin cancers, being attributed to over 99% of non- melanoma skin cancers and over 95% of melanomas, and there are two kinds of UV radiation: o UVA is deeply penetrating to the dermis and causes genetic damage to cell and immune suppression o UVB penetrates into the superficial epidermis and has roles of cell damage and sunburn in melanomas  HPV is a globally common virus with over 100 subtypes, around 14 of which can cause cancer – 70% of cervical cancers are associated with type 16 and 18 HPV infection o Chronic infection can lead to development of precancerous lesions that become malignant when the invade across the basal membrane and become disseminated o Two proteins of HPV that contribute to carcinogenesis are E6 which inhibits the p53 tumour suppressor protein (mentioned above), and E7, which inhibits the RB (retinoblastoma) protein which normally prevents cell division by blocking transcription factors  Gene expression is the process whereby the genetic information encoded in the DNA is converted into a functional product or protein – expression is a complex process that results in over 200 cell types in a typical human body despite containing the exact same DNA molecule o Gene expression also results in differentiation of pluripotent embryonic cells into more specialised mature cells with distinct phenotypes  The central dogma of molecular biology describes the processes of DNA replication, RNA transcription and translation, which essentially result in formation of proteins from DNA  Gene expression can be controlled or regulated at numerous stages of the central dogma – e.g. transcription, translation etc.  During transcription, segments of DNA are transcribed into RNA by an enzyme called RNA polymerase – the DNA consists of distinct sequences and signals that control the start and stop points of transcription o cis-regulating elements are regions of DNA that lie adjacent or neighbouring to the coding region that are involved in regulation of transcription of that nearby gene by allowing positioning of RNA polymerase at the promoter region o trans-acting factors (or transcription factors) are elements that bind to the cis- regulating elements thus influencing gene expression  There are hundreds of transcription factors that have been identified, each of which binds to a particular sequence of DNA – specific combinations of transcription factors are required to activate a particular gene, and the transcription factors themselves are regulated by other factors (e.g. hormones, drugs etc.) o Transcription factors can be regulated by ligand-binding, meaning that a transcription factor can be inactive until bound by a ligand or other element which causes a conformational change that allows binding of RNA polymerase, which then binds to the promoter region of the DNA o These factors are often spatially regulated, meaning that they are expressed in certain tissues or organs, and temporally regulated, meaning that they are expressed during certain stages of development  Enhancers can also be relatively far from the promoter region, and when bound by the transcription factors, they cause bending of the DNA and recruitment of certain proteins to form the transcription initiation complex  Following transcription, there are several post-transcriptional modifications that contribute to stability and function of the RNA transcript and serve as regulatory mechanisms for gene expression  o The synthesised transcript is capped and polyadenylated in order to prevent rapid degradation of the RNA as well as to anchor the molecule to the ribosomes during translation o Intron splicing is a process whereby the introns in the RNA sequence are removed and the exons that encode proteins are ligated to form a coding RNA molecule – a phenomenon called alternative splicing describes that some exons can be included or excluded to form different proteins from a single monocistronic gene (e.g. calcitonin in the hypothalamus and thyroid) o Other mechanisms of regulation include varying rates of transport of mRNA from the nucleus to the cytoplasm, varying degrees of longevity or stability of mRNA and varying rates of degradation of mRNA by ribonucleases  Non-coding RNA (ncRNA) form the majority of the transcription products of the genome (mRNA only forms 3-5% of transcription) – these ncRNA transcripts are often precursors that require further processing to become mature  Some small ncRNA can be involved in gene expression by regulating mRNA translation or by configuration of chromatin o An example is the formation of RNA-induced silencing complexes (RISC) – the small ncRNA can bind to complementary sequences on target mRNA, which guides the RISC to the target mRNA resulting in down-regulation of gene expression o These small ncRNAs are around 22 base pairs in length and are called small interfering RNA (siRNA)  Translation is the process whereby the transcribed RNA sequence is decoded in sets of 3 nucleotides called codons – each codon has a complementary binding site on a tRNA molecule that carries and aligns the matching amino acid o Thus, the function of ribosomes is the alignment of amino acids based on mRNA sequence and catalysing the formation of peptide bonds between the amino acids to form a polypeptide chain o The genetic code is said to be redundant, meaning that a given amino acid can be encoded by several codons  There are several mechanisms by which translation of an mRNA transcript can be regulated: o Physical regulation or blockage of ribosomal machinery from attaching to the mRNA, thus preventing commencement of translation (e.g. RISC) o Initiation factors are those that bind to and assist in the assembly of the ribosomal complex in order to initiate translation – various proteins control the availability of these factors to control translation of some genes  o tRNA heterogeneity and codon usage bias is a phenomenon that describes differences in relative abundance of certain tRNA molecules in different tissues, as well as preferential use of specific codons, results in regulation of the rate to translation  In other words, if a certain tRNA molecule is relatively rare and the mRNA molecule being prescribed has a high number of codons specific to that rare tRNA molecule, then the rate of translation will be slow  Similar to post-transcriptional modifications, there are numerous post-translational modifications that occur after translation of the protein – these modifications are important for achieving functional diversity of proteins, guiding protein folding and regulating protein activity  Molecular chaperone proteins are important factors in the correct folding of a protein following translation, which is important for correct protein function – incorrect folding of a protein can lead to degradation or loss of function of the protein  Post-translational modifications include: o Covalent addition of functional groups to the protein such as methylation, phosphorylation, alkylation etc. which often occur on particular amino acid residues and function to control the activity of the protein o Proteolytic cleavage of regulatory subunits that can either activate or inactive the protein o Degradation of entire proteins by proteasomes can be done in some cases, such as misfolding of the protein  Epigenetic regulation is defined as alterations that can control gene expression without any changes to the DNA sequence – epigenetic alterations can be hereditary and can have a role in carcinogenesis  A prime example of epigenetic regulation is DNA methylation, which involves addition of a methyl group primarily to cytosine residues in CpG islands; the addition of a methyl group results in restricted access of transcription factors to promoters, thus inhibiting transcription o CpG islands are regions of DNA that have a high concentration of C-G residues  Histone acetylation is another example of epigenetic modification mediated by various enzymes such as histone acetyltransferases, histone deacetylases etc. o Acetylation is associated with less condensed and transcriptionally active euchromatin; whereas deacetylation is associated with more tightly wound and less active heterochromatin o Cancer cells often overexpress histone deacetylases (HDAC), leading to hypoacetylation of chromatin and reduced transcription – HDAC inhibitors can be potentially have a role in cancer therapeutics by inducing cell cycle arrest and apoptosis  Cancer is a disease of dysregulated gene expression that results in increased proliferative signals and decreased apoptotic signals- these alterations can occur on any level of gene expression  As mentioned before, the two main groups of genes involved in carcinogenesis are oncogenes and tumour suppressor genes – accumulations of mutations on these genes that lead to increased survival and loss of proliferative control results development of cancers  Gene expression profiling is a process whereby the levels of mRNA are detected in cancer cells in order to assess the transcriptional activity or total gene activity of the cell o This process can be important in determining molecular targets for treatment, predicting prognosis of the patient and understanding the underlying molecular mechanisms in the tumour o Given the heterogeneity of cancer, gene expression profiling allows stratification of a population into subgroups based on the underlying mechanism of carcinogenesis,  Tumour heterogeneity is a factor that adds a layer of complexity to understanding and treating cancers: o Inter-tumour heterogeneity occurs between patients such as when one individuals breast cancer consists of cancer cells with different morphology and/or gene expression profile to another individual o Intra-tumour heterogeneity is when a single tumour consists of different cells with different morphologies and gene expression profiles  This phenomenon makes treatment of cancer more difficult, as one therapeutic agent may inhibit one cancer cell strain but not another  Using colon cancers as an example, there are several genes and signalling pathways that have been identified as having a role in progression of cancers o Colorectal cancers have historically been subdivided into those associated with microsatellite instability and those more associated with chromosomal instability o Defective pathways involved in CRC development include RAS-MAPK, PI3K and TGF-β (oncogenes) and p53 and DNA mismatch repair systems (tumour suppressor genes) o More recent studies observed the effect of overexpression of WNT signalling and miRNA activity in oncogenesis and can serve as useful biomarkers, prognostic indicators or even therapeutic targets in CRC treatment  One process that was found to be involved in transition of an invasive carcinoma to metastasis is epithelial-mesenchymal transition (EMT), whereby epithelial cells lose their polarity and adhesion to cells and gain migratory and invasive properties  o EMT is a naturally occurring process in tissue repair, but is also involved in initiation of metastasis o A potential therapy may involve blocking the phenotypic transition of epithelial tumour cells into mesenchymal cells without interfering with normal tissue healing  An example of a cancer where the gene expression profile has improved diagnosis, prognosis and understanding of the molecular processes involved is breast cancer o Breast cancer is a heterogenous group of cancers with different features and expression profiles – at least 5 major subtypes of breast cancer have been identified based on their molecular cause, responsiveness to therapy etc. o Breast cancers are essentially caused by changes in gene expression that lead to aberrant cell growth, as well as changes in polarity and cell-cell communication  As mentioned previously, neoplasia refers to the excessive and uncontrolled proliferation of cells that persists even in the absence of initial growth stimuli – neoplasms can either be defined as benign or malignant depending on their capacity for invasion and metastasis  A benign neoplasm is one that lacks the ability to invade other tissues and metastasise to different body regions – these are generally innocuous and can be removed surgically but can cause problems depending on the site of growth (e.g. meningioma can cause headaches, seizures etc.) o The macroscopic appearance of a benign tumour is that of a generally rounded shape that is well-circumscribed from surrounding tissue and often encapsulated with fibrous tissue o The microscopic features include relatively uniform cells with a low mitotic rate and well-differentiated (low anaplasia)  Malignant tumours are those with the capacity to invade adjacent tissues and metastasise to different body areas; they are aggressive in their growth and are associated with higher morbidity and mortality than benign tumours o The macroscopic features of a malignant tumour include a crab-like infiltrative shape, not encapsulated and hard and fibrous texture o The microscopic features include pleomorphism, anaplasia (poor differentiation), hyperchromasia (overactive nuclei), and abnormal mitotic figures  Invasion and metastasis are two hallmarks of cancer – in other words, benign tumours are not cancers; malignant tumours are o Invasion and metastasis are huge contributing factors to mortality and morbidity associated with cancer, and the extent of invasion/metastasis can be used as prognostic indicators  Essentially, invasion is defined as the ability of a tumour to disseminate from the boundaries of the organ or origin and cross into foreign tissue; metastasis is the ability of a secondary tumour to be established at a region of the body distant to the primary organ o Both invasion and metastasis are complex, multistep pathways where different processes contribute to carcinogenesis  There are four main steps involved in the development of invasion – altered cell adhesion, ECM dissolution, altered interactions between cells and the ECM and the ability of locomotion  Altered cell-cell adhesion: normally, tight adhesions between cells lead to various signal transductions that regulate growth, maintain differentiation and prevent cytoskeleton remodelling – this is called contact inhibition o In cancers, adhesion molecules, such as E-cadherin are downregulated so that cells dissociate from one another and lose this contact inhibition  Matrix dissolution: the extracellular matrix needs to be degraded for invasion to occur – cancerous cells can induce secretion of proteases (MMPs, heparinases, serine proteases) to degrade the matrix o The ECM consists of a basement membrane and interstitial matrix – the basement membrane is a dense collection of collagen, glycoproteins and proteoglycans; the interstitial matrix is a loose matrix of water, fibrous proteins and adhesive glycoproteins o Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteases that are involved in embryogenesis, growth and tissue repair – they are strongly associated with invasive cancers as they can also physical passage of tumour cells o Tissue inhibitors of MMPs (TIMPs) are a family of four proteins that block the activity of MMPs by ligating to the zinc-binding site of the protein – they are secreted in order to limit tumour invasiveness  Altered cell-matrix adhesion: cells have receptors that bind to ECM proteins along their basal surface that are involved in transduction of inhibitor signals similar to cell-cell interactions (contact inhibition) o Integrins are a family of transmembrane glycoprotein heterodimers that are attached to the intracellular cytoskeleton – integrins are involved in regulating the shape of the cell, as well movement and orientation o In cancers, there is often altered gene expression of integrins – some integrin subtypes almost always promote carcinogenesis (e.g. αVβ3), while others are seen to inhibit certain cancers (e.g. α2β1 inhibit breast cancer)  Locomotion: the locomotion or migration of cells is the last step in invasion, and is a complex process involving various signals and proteins that produce changes in the cytoskeleton (e.g. loss of contact inhibition, MMPs etc.) o Migration of cells can either occur as single cells (mesenchymal) or as groups of cells (collective) – both are slow processes in which the leading edge of the moving cell/cells attaches to the ECM, and contracts to propel the mass forward o Collective migration is advantageous for survival of tumor cells, as there is more efficient movement, protection of underlying cells from immune recognition, higher concentration of autocrine factors and passive transport of non-motile cells o Another mechanism of locomotion is amoeboid migration, in which the movement of the cell is mainly dependent on cytoskeletal remodeling that allows the cell to squeeze through components of the ECM without degrading it – this can explain the poor response of cancers to anti-MMP therapies as cells can switch between amoeboid locomotion and either collective or mesenchymal locomotion  After invasion of the extracellular matrix, tumour cells can access blood vessels, lymphatics and body cavities that can lead to distant spread of the tumour cells, result in metastasis o The three main ways a tumour can metastasise are: direct seeding of body cavities via penetration of the membrane, dissemination via lymphatic (esp. in carcinomas) and haematogenous spread (more often in veins due to thinner walls)  Metastasis is a very complex process that requires a number of process to occur and a number of signature genetic changes to facilitate these processes – it is estimated that around 0.01% of invasive tumours successfully metastasise into the secondary neoplasm o Primary tumours are genetically unstable and produce a heterogenous mass of multiple cell clones; some mutations developed in early carcinogenesis can contribute to metastasis, but other mutations are needed to form a metastatic phenotype  The first step of metastasis is intravasation, which is the tumour cells invade the blood vessels by penetrating through the basement membrane – this requires interactions between integrins and the extracellular matrix, and proteolytic enzymes that penetrate the ECM  Following intravasation, the tumour cells enter the blood stream to be transported to distal areas (vascular dissemination) – very few cells survive in the bloodstream due to the presence of immune cells and mechanical stressors o The tumour cells have a survival advantage when aggregated with platelets and fibrin; tumour cells can secrete procoagulant and platelet activating factors, as well as increased expression of adhesion molecules o Tumour-platelet thrombi shield tumour cells from mechanical stress and immune cells, as well as facilitate extravasation  Extravasation is the arrest of circulation of a tumour cell, adhesion to the endothelium of the blood vessels and egression through the basement membrane – this is facilitated by expression of adhesion molecules on the tumour-platelet thrombus o Extravasation requires interactions between integrins and ECM, as well as proteolytic enzymes to breakthrough the matrix, similar to intravasation  The final step of metastasis is colonisation, which is the process by which the cancer cells adhere to and form a growth on a tissue or organ – the site of metastasis is often the first capillary bed (e.g. lungs) but often differs between cancer cells o The ‘seed and soil’ hypothesis describes that certain cancers (seeds) thrive selectively in certain environmental niches or body regions (soil) – e.g. cancer cells expressing CD44 often deposit in lymph nodes due from interactions with the CD44 receptor (hyaluronate); breast cancers expressing CXCR4 often migrate to bone marrow, where the ligand SDF1 is highly expressed o An important process that is essential for colonisation is angiogenesis – stimuli for angiogenesis are induced by cancer cells as a normal response to hypoxia (HIF-1 & 2), as well as genetic changes called the angiogenic switch that promote angiogenic factors and inhibit anti-angiogenic factors (activation of RAS, MYC; suppression of p53) o Thus, the site of metastasis depends on the tumour cells, the microenvironment of the host tissue and the circulatory patterns surrounding the host tissue  Cancer is a heterogenous disease, so there is little to no chance of a single treatment for all cancers – therapies can target various pathways and novel molecules of invasion and metastasis (e.g. MMP inhibitors, VEGF inhibitors etc.)  Signal transduction is a process that is normally important to cell activity and survival in response to changes in the environment – it is defined as process in which a cell converts an extracellular signal into a cellular response  Signal transduction is involved in cellular communication, homeostasis and interactions between the cell and the environment, resulting in functional changes such as apoptosis, proliferation, metabolism, angiogenesis migration etc.  Signalling transduction pathways consist of several essential components: o A ligand is a compound that acts as an extracellular signal to initiate the signal transduction cascade – these can include growth factors, neurotransmitters, hormones, cytokines etc, o Ligands bind to specific receptors on the target cells – receptors are ligand-specific and can either occur on the plasma membrane or within the cell (ligand dissolves through membrane into the cell)  o Receptors often have intracellular domains that result in activation of a transduction cascade, which often involves the use of secondary messengers (e.g. cAMP, Ca2+ etc.) o The target is the active process which is altered in order to produce the desired effect, and this can be an enzyme, cytoskeletal protein, gene regulators etc. o The final component is the response, which varies between different types of signals and target pathways (e.g. altered metabolism, gene expression, cell shape etc.)  Cells often have specific circuits or pathways for different functions and processes (e.g. motility, differentiation, proliferation, survival) – these pathways can often be linked together via certain intermediates  A simple example of a signalling cascade is that of erythropoietin, which is involved in the stimulation of erythropoiesis (production of erythrocytes): o The erythropoietin exists in an inactive monomeric conformation with an intracellular domain that has an attached tyrosine kinase called Janus kinase (JAK-2) o Binding of erythropoietin to the receptor causes a conformational change that results in dimerization of the receptor, which results in phosphorylation of the attached tyrosine kinase JAK-2, which in turn phosphorylates an intracellular secondary messenger called signal transduced and activator of transcription (STAT-5) o STAT-5, which normally exists in a singular form, becomes dimerised when phosphorylated by JAK-2 and consequentially becomes able to cross the nuclear envelope and act as a transcription factor and alter gene expression  An example of a signalling cascade that is often disrupted or mutated in cancers is the EGF- receptor (epidermal growth factor) family, which is also called the HER2 receptor family; these receptors are often over-expressed in breast, gastric and lung cancers  The EGF-receptor family utilise two prominent intracellular signalling cascades to transduce an extracellular signal to stimulate cell proliferation – these two pathways are the RAS/RAF/ERK and the PI3K/AKT pathways  These two pathways can be subject to targeted treatments, as a number of receptor families and cancers utilise these pathways – developed drugs can act as inhibitors that can either bind to and block the receptor, or inhibit the enzymatic phosphorylation of different proteins within the pathway  Stimulation of a receptor (e.g. EGF receptor) results in phosphorylation and activation of the RAS protein, which is mediated by the proteins GRB2 and SOS o RAS catalyses the phosphorylation of RAF, which phosphorylates MEK-1/2, which in turn phosphorylates ERK-1/2 – ERK translocates to the nucleus to promote cell differentiation, proliferation and growth  o Scaffold proteins are involved in the RAS/RAF/ERK pathway by binding to the components of the pathway and keeping them in close proximity, thus allowing the pathway to progress rapidly and efficiently  The RAS/RAF/ERK pathway is one which is relatively common among cancers, and different components of the pathway can be mutated in different cancer subtypes  Several drugs have been developed to target different points along the signal transduction pathway, with the most successful drugs being those that target MEK-1/2  Melanomas are a subtype of cancer that have been relatively responsive to targeted treatment of the RAS/RAF/ERK pathway, since proliferation of melanomas is dependent on B-RAF mutations o While normally, cell proliferation under the control of B-RAF is tightly controlled, some mutations in the B-RAF gene can develop that allow the pathway to be ubiquitously activated, resulting in uncontrolled proliferation of the melanocytes o A B-RAF inhibitor called vemurafenib is a selective inhibitor of constitutively expressed B-RAF kinases and thus blocks the downstream activity of the signalling cascade, preventing cell proliferation  The issue with targeted treatments such as vemurafenib is the development of mutations that result in the progression of the cancer – in the context of vemurafenib and melanomas, this can occur in two main ways: o MEK-dependent progression means the same pathway is being utilised by the cancerous cells (RAS/RAF/MEK/ERK), but mutations downstream of the RAF protein have been acquired that result in loss of efficacy of the drug in preventing cell proliferation o MEK-independent progression refers to the utilisation of a different signal transduction pathway (e.g. PI3K/AKT) in order to promote cell proliferation  AKT is another signalling protein involved in a number of different pathways involved in protein synthesis, cell survival and glucose metabolism – it is a serine-threonine kinase that can be activated by a number of different upstream signalling molecules  An example of a pathway involving AKT is the PI3K/AKT/mTOR pathway, which is involved in preventing apoptosis and promoting cell survival  PI3K (phosphatidylinositol 3-kinase) is an enzyme that consists of two main subunits (p85 and p110), and is activated by a number of different signals (inc. RAS) to phosphorylate a lipid compound called PI(4,5)P2, converting it to PI(3,4,5)P3, also called PIP3 o PIP3 can attach to the PH domain of the AKT protein, which activates the protein to stimulate numerous cell functions (e.g. preventing apoptosis, cell proliferation)  o PTEN is a phosphatase enzyme that acts as a regulatory molecule in this process by catalysing the removal of PIP3 from the AKT kinase, thus inhibiting the pathway  Numerous steps of this pathway can be and are the target for a number of therapeutic approaches – examples of drugs in clinical use include everolimus, temsirolmus (mTOR inhibitors) and perifosine (AKT inhibitor)  Chronic myeloid leukaemia is a prime example of a cancer directly caused by a genetic mutation that causes overactivation of various signalling pathway – the cancer is caused by a translocation mutation between BCR and ABL, resulting in a fusion protein o Translocation of the ABL domain removes the negative control system that downregulates expression of the protein, resulting in constitutive tyrosine kinase activity o The BCR-ABL fusion product stimulates a number of different signalling pathways including JAK/STAT, RAS/RAF/MAPK and PI3K/AKT  A tyrosine kinase inhibitor that was developed to prevent constitutive expression of the BCR- ABL fusion protein is Imatinib, which binds directly to the ATP binding site of the protein and downregulates the overactive proteins  An issue with Imatinib therapy is the development of mutations that confer resistance to the therapy, and these mutations can occur directly on the binding site of imatinib (e.g. T315I), or in different regions that indirectly render the drug ineffective  Hanahan and Weinberg (2000) published an article summarising the ‘hallmarks of cancer’, as shown in the figure – modern research and developments in understanding of the mechanisms that underlie cancer led them to update their article in 2011 to include other hallmarks such as genome instability, dysfunction of cell energetics, avoiding immune recognition and tumour- promoting inflammation  One particular hallmark of cancer is ‘replicative immortality’ in which cells are able to replicate at a seemingly endless rate – this is caused by telomere elongation, in which the ends of the chromosomes (telomeres) are longer and facilitate cell cycle progression o Normal cells often have the capacity to replicate a limited number of times (30 – 50) before entering a state called senescence in which the cell cycle is arrested, and cell division stops – this process is mediated by tumour suppressing genes such as p53 and RB o Most somatic cells have little to no telomerase activity, while increased telomerase activity is seen in 85 – 95% of all cancers  Some normal body cells need unlimited replicative capacity such as germline cells (high telomerase activity) and, to some extent, progenitor stem cells  In the case of normal somatic cells, telomere length decreases over time and with each cell division, and when the length falls below a threshold the cell enters a state called senescence, in which the cell cannot undergo further replication o Over time, senescent cells undergo a process called mitotic crisis, which results in cell death o Cancerous cells have mechanisms that increase telomerase activity, thus enabling the maintenance of telomere length so that the cell has unlimited capacity to divide o Telomerase inhibition offers a potential cancer therapy by stimulating the shortening of telomeres to force the cells into mitotic crisis  The cell cycle refers to the series of events that occur throughout the life of a cell, and consists of two main processes: o nterphase is the phase in which a cell spends most of its lifespan, and include three distinct subdivisions: the G1 phase in which the cell grows and increases its cytoplasmic volume and protein synthesis, the S phase, in which DNA replication occurs, and the G2 phase, in which the cell grows in preparation for mitosis o The mitotic phase is the phase of the cell cycle in which the replicated DNA is divided and separated to produce two daughter cells from a single mother cell – mitosis refers to the nuclear division and cytokinesis refers to the cytoplasmic division  G0 phase is one that lies outside the replicative cell cycle whereby the cell is not dividing or growing, but stays in a quiescent or resting state – cells in G0 phase can either be senescent cells or terminally differentiated cells o Dividing cells can be made leave the cell cycle and enter the G0 phase due to lack of nutrients, absence of growth factors etc. o Cells in G0 can be made to enter the replicative cell cycle via the action of growth factors called mitogens o The inability of a cell to correctly complete the cell cycle will result in the induction of apoptosis of the cell  All cells have a limited capacity of replication, eventually reaching a point where they cannot replicate further – this is due to the activity of the enzyme telomerase, which acts to maintain or increase the length of the telomeres  Normal somatic cells have little to no telomerase activity, whereas germline and stem cells, as well as cancer cells, often have significant telomerase activity  DNA replication is a semiconservative process that involves the use of numerous proteins: o Helicases unwind the DNA double helix to expose two single strands that are used as templates for replication  o RNA polymerase deposits RNA primers on the DNA strand to allow synthesis of a new strand by DNA polymerase, which adds nucleotides to the primers o DNA replication always occurs 5’ to 3’, so one strand can be continually synthesised without interruption (leading strand) while the other is done in fragments called Okazaki fragments (lagging strand)  Synthesis of the lagging strand is essential for the shortening of the telomers, as RNA primers that are deposited along the length of the DNA strand to allow DNA synthesis needs to be removed – in other words, every time DNA replicates the lagging strand results in the loss of a section of DNA at the telomere (end of a chromosome)  Telomeres are repeating sequences of TTAGGG at the end of chromosomes measuring around 10 000 base pairs in length – telomeres play numerous functions in the chromosomes: o They protect the chromosome from degradation or fusion, since free DNA is reactive and unstable o They protect the chromosome from mechanisms that damage DNA and thus trigger apoptosis o They help position the chromosomes in the nucleus o They maintain the length of chromosomes throughout generations of replication  Telomerase is a ribonucleoprotein enzyme that can act as a reverse transcriptase (convert RNA to DNA) – telomerase carries its own RNA template that binds to the lagging strand template and elongate the strand by adding new nucleotides along the length of the strand  A different process that can be used be cancer cells to elongate the telomeres and increase proliferative capacity of the cell is via alternative lengthening of telomeres (ALT) o One example of ALT is called unequal telomeric sister chromatid exchange, in which adjacent telomeres undergo unequal homologous recombination, resulting in one sister chromatid with an elongated telomere (increased proliferative capacity) and one with a shorted telomere (reduced proliferative capacity) o Another example of ALT is homologous recombination-dependent DNA replication, in which a telomere strand invades the adjacent strand and uses the complementary strand as a template for extension, followed by synthesis of a second strand  Aside from telomerase activation and telomere elongation, there are several processes that are important to the oncogenic transformation of a cell – these include, but are not limited to, suppression of cell cycle inhibitors (e.g. p53, RB) and overexpression of signal transduction pathways (e.g. RAS/RAF)  Telomeres do not exist as free open strands of DNA, otherwise they would be subject to defence mechanisms – telomeres form structures such as T-loops and D-loops in order to ensure that the telomere and the chromosome is protected and stable  o T-loops are essentially circles of DNA where the strand loops around it self o D-loops are formed when the overhang strand invades into a homologous strand (telomeres are repeat sequences) and binds to its complementary sequence, thus hiding and protecting the exposed strand  The Shelterin protein complex is a complex of proteins that bind to the repeat sequence of telomeres and are involved in the protection of the telomeres, as well as the recruitment of telomerase enzymes to control elongation of the telomeres o The TRF1 complex is involved in the recrutment with telomerase to regulate extension of the telomeres; the complex includes a protein called tankyrase, which polyA- ribosylates TRF1 proteins from the telomere and allow telomerase to elongate the strand o The TRF2 complex functions to protect the telomere from homologous recombination, non-homologous end joining and promoting the formation of T-loops; TFR2 also prevents activation of DNA damage responses  Congenital dysfunction of the telomeres and/or telomerase enzymes results in a number of inherited defects: o Dyskeratosis congenita is a rare progressive disorder characterised by short telomerases – results in a variable phenotype with typical manifestations including abnormal skin pigmentation, nail dystrophy and leukoplakia, as well as progressive bone marrow failure leading to mortality o Werner syndrome is an autosomal recessive defect of the WRN protein, which is a helicase protein involved in unwinding DNA – defects result in inability of telomerase to access the telomeres, resulting in shortening of the telomeres, premature ageing and increase risk of cancer o Blooms syndrome is another autosomal recessive disorder resulting in genomic instability and increased rates of homologous recombination and sister chromatid exchange – clinical presentation includes short stature and predisposition for cancer  The widespread expression of telomerase in cancers makes them an ideal target for cancer therapy – inhibitors of telomerase and associated proteins have shown good response rates in clinical trials and are more effective in conjunction with other therapies o GRN163L is a 13 base pair oligonucleotide that directly binds to and inhibits the RNA template of the telomerase, as well as binding to the active site of telomerase, thus effectively inhibiting enzyme activity o BIBR1532 is a small molecule inhibitor that binds to the active site of telomerase and inhibits enzyme activity  Cell proliferation is an important process in cancer, and refers to the division of cells into daughter cells – cell proliferation is different from cell growth, with is the increase in the size of the cell, often in preparation for division o Cell proliferation is a tightly controlled process that is determined by extrinsic factors (e.g. nutrient availability, growth factors) and intrinsic factors (e.g. intracellular signalling pathways) o In cancer, the tight control of cell proliferation is compromised, and cells grows and divide uncontrollably  Cells grow and divide in a sequential manner called the cell cycle, which is described earlier – in order to ensure faithful cell replication, there are numerous checkpoints between phases of the cell cycle that either allow progression of the cell cycle or induction of apoptosis of the cell o In phase G1, surveillance mechanisms ensure the genome is viable and not damaged before allowing progression to S phase o In the S phase, when DNA is replicated, the cell cycle halts if the genome is damaged o In phase G2, the cell is only allowed to progress to mitosis if the genome has been fully replicated  During phase G1, the cell is responsive to external stimuli such as growth factors and nutrient availability, and these signals either allow progression of the cell into S phase, or induce exit of the cell into G0 o If the cell cycle progresses or commits to cell division, it becomes unresponsive to external stimuli and either completes the cell cycle or undergoes apoptosis – the point at which the cell commits to cell division is called the restriction point (R point)  One of the key enzymes involved in progression of the cell cycle a family of serine-threonine kinases called cyclin-dependent kinases, of which over 5 have been identified and their role established in the cell cycle o CDKs are often inactive and have little kinase activity when not bound to cyclin  CDK/cyclin complexes are active kinases that can phosphorylate serine or threonine residues on target proteins  There are numerous families of cyclins (cyclin A, B, D, E) and numerous subtypes of cyclin- dependent kinases (CDK1, CDK2, CDK4, CDK6) and each group of these proteins play a role in regulation of different phases of the cell cycle o CDK4 or CDK6 interact with cyclins D1, D2 or D3 to allow progression of G1 phase o Following the restriction point, CDK2, along with cyclins E1 or E2, are responsible for promoting transition of the cell from G1 to the S phase o Entry and progression of the cell into S phase is mediated by CDK2, which interacts with cyclin A1 or A2  o In late stage S phase, cyclin A1 or A2 switches to CDK1 to promote completion of the S phase and progression into G2 o The M phase is initiated and mediated by binding of CDK1 to cyclins B1 or B2  The expression of CDK families is fairly constant throughout the phases of the cell cycle, but the availability of cyclins fluctuates throughout the cell cycle o The cyclin D family is controlled by growth factors and extrinsic signals (thus phase G1 is responsive to environmental signals), but after the restriction point, expression cyclins become controlled by intrinsic signalling pathways  The regulation of the activity of CDK-cyclin complexes is done by a group of proteins called CDK inhibitors (CDKI), of which there are two classes: INK4 inhibitors and Cip/Kip inhibitors  Inhibitors of CDK4 (INK4 inhibitors) are a family of proteins that competitively bind to CDK4 proteins which are important in progression of the G1 phase o There are four INK4 proteins named according to their molecular size – p15 (INK4B), p16 (INK4A), p18 (INK4C), p19 (INK4D) o INK4 proteins act by distorting the binding sites of ATP and cyclin D, thus reducing enzymatic activity and reducing affinity of the enzyme for its substrate cyclin D – therefore INK4 is responsible for regulation of progression of phase G1 o CDK4/6 activation is dependent on cyclin D, which is controlled by growth factors and environmental signals – cyclin D has a short half-life, so constant extrinsic stimulation is required for continuous CDK4/6 activation o When the stimulus is stopped, levels of cyclin D decrease and INK4 proteins mediate the cessation of G1 progression  The Cip/Kip inhibitors can bind all cyclin-CDK complexes and control the progression of the cell cycle from G1 to the S phase – these proteins inhibit all cyclin-CDK complexes with the exception of the cyclin D-CDK4/6 complex, which it promotes o Cip/Kip inhibitors include p21 (Cip1), p27 (Kip1) and p57 (Kip2)  Cip/Kip inhibitors are responsible for controlling the progression of the cell from G1 to S phase by enhancing the progression of G1 and preventing premature entry of the cell into S phase o In early G1, there is little expression of cyclins and a high number of Cip/Kip inhibtors; growth factor stimuli increase the expression of cyclin D, which binds to CDK4/6 o The Cip/Kip inhibitors present in the cell act to inhibit the other cyclin-CDK complexes (thus preventing progression into S phase) and promote the interaction between cyclin D and CDK4/6 (promoting G1 progression) o As the growth stimulus persists, more and more cyclin D-CDK4/6 complexes are formed until all the Cip/Kip inhibitors present are bound to the complex – this means  that the other cyclin-CDK complexes (esp. cyclin E-CDK2) are free from the Cip/Kip inhibitors and are able to progress the cell into S phase  The cyclin E-CDK2 complex is important in the progression of the cell cycle from G1 to the S phase as it phosphorylates (and represses) the retinoblastoma protein (pRB), which is a negative regulator of cell cycle progression o pRB is an essential protein in cell cycle regulation that prevents uncontrolled progression of the cell division – it is named after it was found to be defective in a type of cancer called a retinoblastoma  pRB and retinoblastoma-like proteins (p107 and p130) are structurally similar proteins that are called pocket proteins – they are involved in restricting the G1 to S phase transition via the action of a structural motif called the E2F-binding motif o During early to mid-G1, pRB and similar proteins are hypophosphorylated and are bound to E2F transcription factors, thus effectively inhibiting them o The increased cyclin E-CDK2 activity in late phase G1 stimulates the hyperphosphorylation of pRB and its analogues, causing a conformational change that liberates the E2F transcription factors and allows them to activate the genes required for progression of the cell cycle in S phase (e.g. cyclin E) o E2F is degraded after the cells enter S phase  At the molecular level, the mechanism by which hypophosphorylated pRB represses E2F transcription factors, which is bound to DNA, is by recruiting histone deacetylases – this results in removal of acetyl groups from histone proteins to condense DNA and repress expression  Hyperphosphorylation of pRB prevents the binding of pRB to the E2F transcription factors and enables histone acetylases to acetylate histones, unwrap the DNA and activate gene expression  There are numerous pathways aside from cyclin-CDK complexes that regulate the transition of the cell cycle from G1 to the S phase – MYC is an example of a factor that regulates the cell cycle in response to mitogenic signals; p53 is a protein that is involved in regulating the cell cycle in response to DNA damage  MYC is an important protein that belongs to a family of proteins called basic helix-loop-helix (bHLH) transcription factors that bind to a conserved sequence of DNA present on promoters called the E-box (CANNTG) o MYC dimerises with another transcription factor called MAX and, in response to mitogenic signals, they activate transcription of genes that promote growth and proliferation of the cell  The MYC/MAX complex promotes proliferation by increasing expression of cyclin D2, CDK4, E2F transcription factors and CUL1 (which degrades p27)  o When the mitogenic signal is repressed, expression of MYC is repressed and the MAX associates with another factor called MAD, and these two transcription factors suppress these growth-associated proteins o Overexpression of MYC is seen in over 70% of cancers and promotes uncontrolled cell cycle progression  The regulation of cyclin D1 is another mechanism by which the cell regulates cell cycle progression – there are numerous pathways that converge in the upregulation of cyclin D1 o These pathways include, but are not limited to the MAPK, PI3K, JAK/STAT, NF-κB, Hedgehog, HER2 and Wnt pathways o Some cancers have abnormally increased activity of these signalling pathways, resulting in overexpression of cyclin D1 and uncontrolled cancer cell proliferation (e.g. HER2 gene amplification in breast cancer)  p53 is an important transcription factor in regulating cell cycle progression in response to DNA damage and cell stress o Events such as DNA damage, nutrient starvation and hypoxia result in upregulation of p53, which upregulates the expression of p21Cip1, which either arrests cell proliferation until the damage is removed, or induces apoptosis of the cell o Loss of p53 is among the most common events that occur in cancer, and can occur via inactivating mutations in the gene or by gene deletion  Cancer is characterised by an accumulation of mutations that result in uncontrolled proliferation of cells, insensitivity to growth suppressing signals and self-sufficiency in maintaining growth and proliferation stimuli o Genes that control cell cycle progression and cell proliferation include oncogenes, which normally promote cell proliferation and can be upregulated to increase cell proliferation (e.g. RAS, MYC, EGFR, HER2), and tumour-suppressor genes, which normally prevent cell proliferation but can be repressed or inactivated to allow uncontrolled proliferation of the cell (e.g. p53, RB, CDKI proteins)  Abnormal cell proliferation is an important hallmark in cancer, and can be facilitated by mutations in regulatory proteins involved in the cell cycle  Chemotherapeutic agents can target different mutated or dysregulated components of the cell cycle to target rapidly proliferating cells – these may target both malignant and non-malignant cells that are rapidly proliferating and thus result in a number of side effects (e.g. gastrointestinal tract, bone marrow, hair follicles) o Some of these agents include anti-microtubule agents that inhibit arrangement of the mitotic spindle, alkylating agents that cause crosslinking of DNA to induce apoptosis, antimetabolites that prevent DNA synthesis etc.  Modern advancements in cell cycle understanding has led to the development of drugs that are more targeted towards mutated agents in cancers – an example of this is CDK inhibitors, some of which are in the clinical trial phase of development (e.g. palbociclib, abemaciclib, ribociclib)  Cell death is an important feature of cells that has applications in numerous body processes such as the development of complex tissues and organs in organogenesis (e.g. hands), maintenance of cellular homeostasis (i.e. removing damaged cells) and in many facets of the immune response (e.g. killing infected and self-reactive cells)  There two main processes of regulated cell death, which is a highly controlled physiological process in which cells are eliminated to perform normal biological processes o Apoptosis is referred to as type I cell death and is a normal physiological process involved in maintaining homeostasis in response to environmental stimuli o Autophagy is referred to as type II cell death and often occurs in response to extrinsic stresses such as nutrient starvation and infection; it can also be used to regulate the metabolic needs of the cell by breaking down cell components  Necrosis is an uncontrolled process of cell death in which a physical or chemical insult (e.g. mechanical stress, toxins, hypoxia) causes irreversible damage to the cell and results in dysregulated cell death  There are numerous other types of cell death that are not as universal as those mentioned above: o Anoikis is a delayed cell death associated with build-up of autophagy vesicles o Cornification is a type of cell death specific to epithelial surfaces where a layer of dead keratinocytes is generated on the outer layer of the skin o Pyroptosis and pyronecrosis are two processes specific to the immune system involving the death of infected macrophages and secretion of specific cytokines o Necroptosis is a process of ‘regulated’ necrosis often associated with inflammation o Mitotic catastrophe is cell death due to dysfunction of the mitotic phase of cell division  Apoptosis is a strictly regulated process that can be identified by certain morphological and biological features – apoptosis is an actively controlled process that requires energy to proceed o During apoptosis, the membrane integrity is maintained, the chromatin and nucleus condense, the cytoplasm shrinks as the cell fragments into membrane-bound apoptotic bodies and the mitochondria develop pores o The biological processes involved in apoptosis include alteration of membrane asymmetry and controlled pre-lytic fragmentation of DNA, producing a ladder-like pattern when separated by electrophoresis o Apoptosis is initiated by physiological stimuli and usually affects a single cell, and that cell would be phagocytosed by adjacent macrophages or other phagocytes without evoking an inflammatory response  Necrosis is different to apoptosis in its morphological and biological features, as well as its physiological significance o In necrosis, there is loss of membrane integrity, swelling of the cytoplasm and organelles and total cell lysis without vesicle formation o Necrosis generally does not require input of energy – it is evoked by a non- physiological stimulus that results in loss of ion homeostasis and uncontrolled post- lytic DNA fragmentation, resulting in a smear-like pattern when separated by electrophoresis o This process generally affects a group of cells rather than a single cell, and is often accompanied by a significant inflammatory response due to the leakage of cell components into the extracellular fluid  One more difference between apoptosis and necrosis is the fact that early stages of necrosis can be reversible; cells can display early features of necrosis when exposed to a certain insult but if the stimulus is removed the cells may revert to a normal healthy state o Once a cell commits to apoptosis, it cannot go back  Apoptosis can be induced by extrinsic factors, such as activation of certain receptor signalling pathways, or by intrinsic factors, such as DNA damage – the signalling of apoptosis is mediated by a family of cysteine proteases called caspases o The apoptotic stimulus results in activation in a subtype of caspases called initiator caspases, and these include caspase subtypes 2, 8, 9 and 10 o These initiator caspases result in the cleavage of precursors of a second class of caspases called effector caspases, which include subtypes 3, 6 and 7 – effector caspases then cleave other cellular substrates that perform the processes of apoptosis  The extrinsic activation pathway is mediated by receptor-ligand interactions – an example is the TNF receptor superfamily, which are receptors that contain a domain called the death domain; examples of receptors that fall under this superfamily include TNFR, Fas and death receptor o Ligands that bind to these receptors include TNF, FasL (Fas ligand) and TRAIL  The intrinsic activation pathway is mediated by a cytoplasmic heterodimer consisting of BCL2 and BAX proteins – cellular stresses such as DNA damage or growth factor withdrawal causes liberation of the BAX protein from the dimer, which enters the mitochondria to increase its permeability and release of cytochrome C proteins o The released cytochrome C proteins bind to APAF1 proteins (apoptosis protease activating factor), which contributes to the formation of the apoptosome – the apoptosome then initiates a series of caspase activation cascades that result in apoptosis  Regulation of apoptosis is achieved via an interplay between positive and negative apoptotic modulators  One critical regulator of apoptosis is the BCL-2 family, which was originally named due to its role as an inhibitor of apoptosis in B-cell lymphomas – it was later found to consist of a number of related proteins that have roles in inhibiting and activating apoptosis  The members of the BCL-2 family have been identified by morphological homology – the BCL-2 protein and some of its analogues contain four BCL-2 homology domains (BH domains), as well as a transmembrane domain that is responsible for localisation of the proteins at inner membranes o The main inhibitors of apoptosis include BCL-2, BCL-XL, BCL-1W and MCL-1 and these contain all four BH domains o The BH3 proteins are pro-apoptotic and are so named because the only share a single BH3 domain with other members of the BCL-2 family, and they include proteins such as BID, BIM, BAD, BIK, NOXA and PUMA – these proteins act by blocking the action of anti-apoptotic BCL-2 proteins o The effector proteins are those that actively form pores in the mitochondria and facilitate progression of apoptosis, and these possess three of the four BH domains  Under a survival signal, anti-apoptotic proteins (e.g. BCL-2) predominate in the cytoplasm and effectively inhibit pro-apoptotic proteins (e.g. BAX); also, BH3 only proteins are inactive due to the presence of an inhibitory domain within the protein o An apoptotic signal results in increased expression of BH3 proteins such as BIM, which displace BAX and inhibit BCL-2 proteins, thus liberating pro-apoptotic BIM proteins; also BID is cleaved to form a truncated version of the protein (tBID) o This results in ability of BAX to effectively perforate the mitochondrial membrane to facilitate progression of apoptosis – the process is called mitochondrial outer membrane permeabilization (MOMP)  There are numerous other negative modulators of apoptosis that inhibit various steps in the apoptosis activation cascade: o IAP (inhibitor of apoptosis proteins) block initiator and effector caspase activation – these proteins are themselves inhibited by proteins released from the mitochondria (SMAC, OMI) to allow progression of apoptosis o Pro-thymosin α (Pro-T) inhibits the initiation of the caspase pathway by inhibiting formation of the apoptosome o The E1B protein resembles BCL-2 in its function and acts by binding to and inhibiting positive modulators of apoptosis such as BAK and BAX  Positive modulators of apoptosis are responsible for the induction and progression of apoptosis  o Cytochrome C and the APAF1 proteins are responsible for the formation of a protein complex called the apoptosome, which initiates a cascade of protein substrate cleavages involving effector caspases o Numerous proteins are also involved in chromatin condensation and degradation of DNA, such as the apoptosis inducing factor and endonuclease G – both of these proteins are released from the mitochondria upon permeabilization of the membrane o Granzyme A is a serine protease that is secreted into infected cells by cytotoxic lymphocytes (NK cells, CD8+ cells), and the effect of these granzymes is the induction of apoptosis of the infected cells  Other positive modulators of apoptosis include the tumour suppressor genes (p53, retinoblastoma) which, in addition to regulating progression of the cell cycle, enhance the expression of pro-apoptotic proteins – p53 also reduced expression of anti-apoptotic proteins  Various signalling pathways are responsible for inhibition of pro-apoptotic proteins – one primary signalling pathway is the PI3K/AKT pathway, which phosphorylates BAD and other proteins that promote apoptosis, as well as activate pathways that act to inhibit apoptosis and promote cell survival o Other signalling pathways that act as negative modulators of apoptosis include NF-κB, ERK and PKC  Thus, whether or not a cell undergoes apoptosis or continues to survive depends on the balance of pro- and anti-apoptotic proteins in the cell  Numerous methods of studying and observing apoptotic cells are utilised by identifying presence of caspase proteases, alterations in membrane integrity and fragmentation of morphological changes in DNA  One means by which an apoptotic cell can be identified is by the activity of caspase 3 enzymes in the cytoplasm, which are responsible for mediating the progression of apoptosis – caspases are cysteine proteases that cleave a specific amino acid sequence (DEVD sequence) o One method of detecting the presence of these proteins is by adding a caspase 3/7 substrate attached to a dye; if the cell contains caspases, they will cleave the DEVD sequence of the reagent and allow the dye to enter the nucleus and stain the DNA o A similar assay can be conducted by binding a DEVD substrate to a Luciferin enzyme which, when cleaved, produced luminescence and changes the colour of the cell suspension o Western blotting can also be used to identify the presence of the cleaved products of caspase 3/7 activity, which indicates that the cells are undergoing apoptosis  Another marker of apoptosis that can be used to identify apoptotic cells is the presence of phosphatidyl serine on the outer side of the plasma membrane – as mentioned before, alteration of the asymmetry of the plasma membrane is a biological feature of apoptosis o A fluorescent assay involving the binding of fluorophore-labelled proteins to external phosphatidylserine to indicate if a cell is undergoing apoptosis o Different distinct surface markers can also be used to detect cells that are undergoing necrosis  DNA fragmentation can be performed via electrophoresis to identify the survival state of the cell – apoptotic cells will have a ladder-like pattern produced, whereas surviving cells will have a single band of large DNA o Necrotic cells will have a distinct smear pattern due to random degradation of DNA  The TUNEL assay (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) is another method for detecting apoptosis, and it does this by adding dUTP nucleotides that have been labelled with a marker – the enzyme terminal deoxynucleotidyl transferase adds these nucleotides to the 3’ end of DNA fragments, producing a detectable label o This assay indicates apoptosis as it detects the presence of excessive DNA fragmentation, which is often associated with cell death  Confocal microscopy can capture nuclear condensation and/or fragmentation, which is also often associated with progression of apoptosis – this process can also be used to observe nuclear swelling, which occurs in necrosis  Apoptosis is normally a strictly regulated process, and dysregulation of apoptosis can result in cancer due to increased resistance of cell death and subsequent cell proliferation  There are many mechanisms and cellular pathways that can be overcome leading to resistance of apoptosis o Some extrinsic mechanisms of apoptosis resistance include loss of pro-apoptotic signals, viral interference with normal apoptosis (e.g. HPV inhibition of p53) o Intrinsic mechanisms include overexpression of anti-apoptotic factors (e.g. MYC, BCL-2) or inactivation of pro-apoptotic factors (e.g. p53, RB, BAX, caspases)  Apoptosis induction offers a potential target for cancer therapy, as it involves utilisation of cellular machinery to reduce cell proliferation, and can thus downregulate inflammation and tissue damage o The issue with this approach is the fact that cancerous cells are resistant to apoptosis, and so the underlying mechanism that causes this resistance needs to be identified in each case and overcome, and there are various mechanisms by which this can be achieved  o A developing drug that can be used to target the apoptotic pathway in cancer is venetoclax – this drug acts by inhibiting the anti-apoptotic BCL-2 protein, and promotes liberation of pro-apoptotic proteins such as BIM and BAX to perforate the mitochondrial membrane and facilitate apoptosis  Cancer is a complex and multistep process that involves an interplay of a number of causative and contributing factors including radiation, chemicals, hormones, hereditary predispositions and infections (viral, bacterial)  Viruses are defined as non-living entities that are able to infect and integrate into a host cell and utilise cellular machinery to produce more viral particles – therefore it is difficult to culture viruses in vitro; chicken embryo and other cell cultures can be used to grow some viral strains o Viral carcinogenesis is caused by the insertion of viral genes into host DNA and expression of viral proteins to promote cell proliferation and oncogenic transformations  The typical structural features of a virus include a relatively small size, genetic material (which can be single or double stranded DNA or RNA), characteristic shapes and may or may not consist of a viral envelope o The genetic information is enclosed in a protein capsid, which are collectively called the nucleocapsid o Some viruses are enclosed in a viral envelope, which consist of ligands the mediate cell attachment and infection, and sometimes a membrane that is derived from a host cell o Viruses often differ in their shape and structure, varying from simple helical or polyhedral structures to more complex and irregular morphologies  In the case of viruses with a DNA genome, replication occurs in the nucleus of the host cell, whereas with RNA viruses (retroviruses), replication occurs in the cytoplasm  The term oncovirus refers to viruses that have the ability to cause cancer, and there are six main viral species that are recognised as being oncogenic in humans – HBV, HCV, certain strains of HPV (16 & 18), EBV, HIV-1 and HTLV-1 o Infections are attributed to around 18% of human cancers, with 12% being from viruses – bacterial infections have also been attributed to human cancer (e.g. H. pylori and stomach cancer  Most viruses exhibit a lytic life cycle, which is essentially a cycle of viral infection of a host cell, replication of virion particles, lysis of host cells and release of the progeny virus particles  Some cells exhibit a latent life cycle, in which a virus infects a cell and, instead of expressing structural proteins to synthesise new virions, integrates into the host genome, changes properties of the cell and may result in transformation of the cell o Some viral transformations can be oncogenic, such as alterations in control of cell growth, cellular adhesion, motility and invasion  Oncogenes are genes that can potentially contribute to the oncogenic transformation of a cell, and are often a mutated form of proto-oncogenes, which are normal genes that are involved in positive regulation of cell growth o Mutations that result in overexpression of these proto-oncogenes result in excessive cell growth; oncogenes include proteins such as MYC, ONC, CBL, SRC etc. o Proto-oncogenes are denoted with a ‘c-’ suffix – e.g. c-MYC, c-ONC, c-SRC o Some oncogenes can be transmitted via viral integration into the genome, and these are called viral oncogenes and are denoted with a ‘v-’ suffix – e.g. v-MYC, v-ONV, v-SRC  Normal proto-oncogenes can be activated to become oncogenic by several ways – these include gene amplification resulting in overexpression of the gene, mutations or viral insertions affecting the regulatory region of DNA that controls expression of the proto-oncogene, and chromosomal translocations that result in upregulation of a proto-oncogene  Tumour suppressor genes, formerly called anti-oncogenes, are essentially those responsible for maintaining nor