Tumour Biology Summary Mikulits PDF

Tumour biology Summary Mikulits Overview of the lecture Epidemiology, risk factors and prevention of cancer -> DNA damage and genomic stability Oncogenes and tumour suppressors Tumour virology and cancer models Tumour heterogeneity Cancer cell signalling Tumour mi...

Tumour biology Summary Mikulits Overview of the lecture Epidemiology, risk factors and prevention of cancer -> DNA damage and genomic stability Oncogenes and tumour suppressors Tumour virology and cancer models Tumour heterogeneity Cancer cell signalling Tumour microenvironment – inflammation and angiogenesis Drug resistance and biomarker of cancer 1 LECTURE 1: EPIDEMIOLOGY, RISK FACTORS AND PREVENTION OF CANCER 1.1 WHY TO KNOW TUMOUR BIOLOGY Cancer development during lifetime of men and women Cancer represents a major health burden and affects every second man and one out of three women during their lifetime. In Austria, similar to other Western Countries, cancer is the second most frequent cause of death. Melanoma patient with end-stage metastatic disease. Response to targeted therapy for 15 weeks is followed by therapy failure after 23 weeks of treatment. After surgical resection of the melanoma, metastases occurred which regress after systemic therapy. However, due to therapy resistance, metastases relapse and spread to distant organs. 1.2 WHY NAME „KREBS“ OR „CANCER“? „Krebs“is derived from the old greek word karkínos which designates both the animal „crab“ (Krebs, Krabbe) and the disease (karkínos -> carcinoma). The Latin word for crab/Krebs is „cancer“. The term karkínos was first used for the disease by Hippocrates 460 – 370 BC Celsus (30 BC) translated the greek term into the latin „cancer“. Why Name Tumour? Galen (160 AD) used the word oncos (greek for swelling) to describe tumours (latin „tumere“ = swell) Tumour (Geschwulst) Tumour is a more general term and includes benign neoplasia and malignant neoplasia (= cancer) 1 1.3 HISTORY OF CANCER Cancer is known since thousands of years Egyptians describe surgical removement of tumours (1600 BC); papyrus Ebers) Hippocrates (460 – 370 BC, referred to as the "Father of Medicine“, realized that (i) cancer is a systemic disease affecting the whole body, and not just a specific organ; (ii) a cancer cure can only be achieved by rebalancing the whole organism through a multidisciplinary, holistic approach, and not just by eradicating the tumour. 1.4 CANCER STATISTICS (2020) Global cancer incidence (new cases) is 19,2 million cases Global cancer prevalence (5-year survival) is 50,5 million cases Global cancer mortality (death by cancer) is 9,9 million cases 1.4.1 Compare with cancer statistics 2012 Global cancer incidence (new cases) is 14,1 million cases Global cancer prevalence (5-year survival) is 32,6 million cases Global cancer mortality (death by cancer) is 8,2 million cases 1.4.2 Cancer Incidence and Mortality In less developed countries, higher incidence and mortality of certain cancer types due to: o Less access to health care o Higher rates of cancer-causing infections (HBV, HPV) o Less cancer prevention In well developed countries, higher incidence and mortality of certain cancer types due to: o Aging of population o Obesity o Higher exposure to risk factors 1.4.3 Cancer in Austria – 2020 Notion: e.g. prostate cancer shows high incidence but rather low mortality indicating less aggressiveness (age-related incidence) 2  e.g. lung cancer shows high incidence and high mortality indicating high aggressiveness 1.4.4 Key Facts on Cancer 2020 The most common cancers are breast, lung, colon and prostate cancer Around one-third of deaths from cancer are due to tobacco use, obesity (body mass index > 25), alcohol consumption, low fruit and vegetable intake, and lack of physical activity Cancer is a leading cause of death worldwide, accounting for nearly 10 million deaths in 2020 Cancer-causing infections, such as human papillomavirus (HPV) and hepatitis, are responsible for approximately 30% of cancer cases in low- and lower-middle-income countries Many cancers can be cured if detected early and treated effectively 1.4.5 Environmental factors are estimated to contribute to 90-95% of cancer cases. Diet is the most significant environmental risk factor, accounting for 30-35% of cancer cases. Tobacco is the second most significant risk factor, contributing to 25-30% of cancer cases. Infections are also a major risk factor, accounting for 15-20% of cancer cases. Obesity and alcohol are additional risk factors, contributing to 10-20% and 4-6% of cancer cases, respectively. Other environmental factors such as exposure to radiation, pollution, and certain chemicals are also linked to cancer development. The genetic predisposition to cancer is estimated to be 5-10%. In familial cancer (5- 10%), the first mutation is inherited, while subsequent mutations are somatic. In sporadic cancer (90-95%), all mutations are somatic. 1.4.6 Types of cancer most strongly associated with environmental factors: Testicular cancer: 8.6% Thyroid cancer: 8.5% Laryngeal cancer: 8.0% Multiple myeloma: 4.3% Lung cancer: 2.6% Colorectal cancer: 2.5% Kidney cancer: 2.5% Prostate cancer: 2.2% Melanoma: 2.1% Breast cancer: 1.8% 1.4.7 Familial Cancer Syndromes Because of an inherited mutation, patients have increased risk to develop tumours at a relatively early age Most hereditary cancers are associated with a "germline mutation" (entered the zygote) and is present in every body cell 3  Most familial cancer syndromes follow autosomal dominant inheritance in which the patient's first degree relatives (children) have a 50% risk of carrying the causative mutation The penetrance of the disease, i.e. the likelihood of a mutation carrier developing a malignant tumour during lifetime is e.g. about 80% to 90% for HNPCC (Lynch syndrome) Screening is required to identify mutation carriers Risk patients require systematic surveillance 1.4.8 Genes and Environment in Cancer Development However, some researcher reported that the majority of cancer is mainly due to „bad luck“ the lifetime risk of cancers of many different types is strongly correlated with the total number of divisions of the normal selfrenewing cells maintaining that tissue's homeostasis Only 30% of cancer risk among tissues is attributable to environmental factors or inherited predispositions The majority (70%) is due to "bad luck," that is, random mutations arising during DNA replication in normal, noncancerous stem cells 1.4.9 Rising Cancer Incidence with Increasing Age Cancer incidence and death are increasing in absolute numbers Age-adjusted incidence shows no general rise of most cancers Increase in cancer incidence and death is mostly due to increased life expectancy Yet, some cancers do (e.g. liver cancer) 1.4.10 Life Expectancy (2020) Geographical differences in cancer incidence might be due to different life expectencies Age-standardized models are required for comparison (e.g. age-standardized rate is a summary measure of the rate that a population would have if it had a standard age structure) 1.4.11 Rising Incidence of Liver Cancer > 2025 Liver Cancer: Liver cancer is the 6th most diagnosed cancer globally. It is the 3rd leading cause of cancer-related deaths. Approximately 850,000 new cases of liver cancer are diagnosed each year worldwide. Hepatocellular carcinoma (HCC) is the most common type of liver cancer, accounting for 75-85% of cases. The global incidence of liver cancer is expected to continue rising in the coming years. This is primarily due to the increasing prevalence of NASH, which is driven by obesity and other lifestyle factors. By 2025, it is estimated that there will be 1 million new cases of liver cancer worldwide. Non-Alcoholic Steatohepatitis (NASH): NASH is a liver disease characterized by inflammation and fatty buildup in the liver. It is a major risk factor for liver cancer. The progression of NASH can lead to fibrosis, cirrhosis, and ultimately liver cancer. 4  The prevalence of NASH is increasing due to rising rates of obesity, diabetes, and metabolic syndrome. The Progression of Liver Disease: The Link Between NASH and Liver Cancer: The chronic inflammation and damage caused by NASH can lead to the development of liver cancer. The risk of liver cancer increases as NASH progresses to fibrosis and cirrhosis. Healthy liver: A normal liver with no inflammation or fatty buildup. Steatosis: A condition where there is excessive fat accumulation in the liver cells. NASH: Steatosis with inflammation and liver damage. Fibrosis: The formation of scar tissue in the liver. Cirrhosis: Severe liver damage with extensive scarring. 1.5 RISK FACTORS OF CANCER Environment, exogenous factors: o Nutrition o Obesity (BMI > 25) o Smoking o Air pollution o Carcinogens (occupational) o Ultraviolet (UV) radiation o Radioactivity (X-ray) o Viruses o Bacteria (Helicobacter Pylori) Endogenous factors: o Aging o Genetic predisposition 1.5.1 Risk of Cancer Affected by Cell Division The risk of tumour development is affected by the process of cell division and resulting cell mass/body weight Mouse: 1011 cell divisions per lifetime; human: 1016 cell divisions per lifetime -> increased risk for human Estimation: Bumblebee bat (smallest mammal) has a weight of 1,5 g (estimated life span 5 years) while the blue whale (largest mammal) has a weight of 1,3 x 108 g (life span 80 years) suggesting that the whale has 106- fold higher risk of cancer compared with the bat (109 reduced to 106 by the 103-fold higher rate of metabolism of the bat) 1.5.2 Cancer Incidence and Carcinogens Exposure to a carcinogenic insult rather than the age at the carcinogenic exposure determines the tumour development 5  Development of mesothelioma: Tumour of the mesodermal lining of the abdominal organs and the lungs (pleura, peritoneum, pericard), induced by exposure to asbestos 1.5.3 Infection Disease and Cancer The graph shows a significant decrease in mortality rates over the century, with several notable milestones: 1900: The death rate for infectious diseases was high, exceeding 1000 per 100,000 population per year. 1918-1919: A major influenza pandemic caused a sharp increase in mortality rates. 1920s: The death rate began to decline, likely due to improvements in public health measures, such as sanitation and water chlorination. 1930s: The introduction of penicillin and other antibiotics led to further reductions in mortality rates. 1940s: The passage of the Vaccination Assistance Act contributed to the decline in infectious disease deaths. 1996: The death rate for infectious diseases had decreased to below 100 per 100,000 population per year. 1.6 HOW CAN CANCER BE PREVENTED 1.6.1 Smoking Cigarette smoke releases over 5.000 chemicals Carcinogens damaging DNA Polycyclic aromatic hydrocarbons, N-nitrosamines, aromatic amines, aldehydes etc Smoking is the leading cause of lung cancer (ca 70%) Other cancers caused by smoking include mouth, pharynx, nose and sinuses, larynx, oesophagus, liver, pancreas, stomach, kidney, ovary, bladder, cervix, and some types of leukaemia 1.6.2 Obesity Overweight or obesity, defined as a body mass index (BMI) > 25 kg/m2 is associated with increased morbidity and mortality worldwide Major risk factor for major diseases, such as heart disease, stroke, diabetes Obesity is associated with an increased risk of cancer 2 billion adults affected globally (1/3 of world population) 6  Obesity rates rise dramatically due to massive worldwide shifts in diet and physical activity patterns 40% of all cancers attributed to obesity Breast, colorectal cancers, endometrial, liver, pancreatic, gallbladder, ovarian, thyroid, multiple myeloma, and renal cancers are attributed to obesity Obesity-related cancer higher in women compared to men 1.6.3 Dietary Patterns Western diet associates with an increased risk for colorectal cancer, pancreatic cancer, breast and prostate cancer etc Western diet is o high in fat and sodium (high in saturated fats) o excess carbohydrates/sugar by cookies, cakes and candies o high calories o low in fruits and vegetables o low in fiber Western diet is mainly present in fast food High risk of cardiovascular disease, diabetes, obesity 1.6.4 Calorie Restriction Calorie restriction regimen results in reduced levels of several hormones, growth factors and cytokines, leading to decreased growth factor signalling, fewer vascular perturbations, and decreased inflammation These responses to calorie restriction result in decreased cancer risk 7  Translation of mechanistic lessons learned from experimental models to human beings is important (see obesity and risk for cancer) 1.6.5 Dietary Factors Dietary factors which protect against cancer and DNA damage Anti-oxidants such as vitamin A, C and E in fruits and vegetables act as trapping agents of free radicals. Vitamin C essential for immune system and neutralization of carcinogenic nitrosamines and more Phytochemicals such as flavonoids, polyphenols, indoles, terpenes and others are present in cruciferous vegetables, garlic, onions, soy, green tea and many other plants. They stimulate the immune system, reduce inflammation, trigger apoptosis and neutralize many carcinogens. E.g. resveratrol (red wine, red grapes) and curcumin (Curcuma) 1.6.6 Vaccination against HPV Infection with Human Papilloma Virus (HPV) occurs in nearly all sexually active people. Around half of these infections are with a high-risk HPV types HPV can infect both males and females Two of these high-risk HPV types, HPV16 and HPV18, are responsible for most HPV- related cancers Both men and women can become infected with HPV and develop HPVcaused cancers HPV-related cancers include cervical cancer, oropharyngeal cancers, anal cancer, penile cancer and vaginal cancer HPV vaccines prevent infection with disease-causing HPV types, preventing many HPV-related cancers and cases of genital warts Vaccination is recommended to be given to preteens 1.6.7 Vaccination against HBV Chronic Infection with Hepatitis B Virus (HBV) is a leading cause of liver cancer (hepatocellular carcinoma; HCC) HBV infection and chronic inflammation leading to cirrhosis is a major risk factor for HCC Globally, about 250 million patients with chronic HBV infection Vaccination is recommended for all newborns, children up to 18 years of age, and all adults at higher risk for infection 1.6.8 Reduce Damage by UV Radiation Protection against UV radiation of human is of high relevance due to reduced hair growth/density. UV photons generate pyrimidine dimers which are mutagenic (e.g. T-T dimers). 8  Use sunscreens containing organic or inorganic compounds blocking UVA1, UVA2 and UVB radiation Chemical filters in sunscreens are aromatic compounds that absorb UV radiation, resulting in excitation to higher energy states. When these molecules return to their ground states, the result is conversion of the absorbed energy into lower-energy wavelengths, such as infrared radiation (i.e. heat) 1.6.9 (Familial) Breast Cancer Who is at increased risk of breast cancer? o Individuals with strong family history of breast cancer o Genetic predisposition, such as an inherited mutation in BRCA1/2 or other breast cancer-related genes o Breast biopsy with atypical cells Cancer prevention programs recommend that women at high risk get a mammogram (X-ray picture) and breast MRI (magnetic resonance imaging) every year starting at age 25 to 40, depending on the type of gene mutation and/or youngest age of breast cancer in the family. Austria: Patient at high risk (e.g. BRCA1/2 and others) annual MRI > age of 25 and mammogram > age 35, sonography whenever; offering Prophylactic Bilateral Mastectomy (PBM). Österreichische Brustkrebs-Früherkennungsprogramm starts at age of 40 (mammogram every 2 years). 1.6.10 Blocking Aging? Cancer is becoming an ever more important health burden worldwide in aging population. In both cancer and aging, the underlying mechanism is the time dependent accumulation of cellular damage. Cancer and aging may seem like opposite processes - cancer cells have the “gain of function and fitness” whereas aging cells are characterized by a “loss of function and fitness” Cancer and aging do share many common characteristics such as e.g. genomic instability and telomere shortening (yet in cancer with telomerase activation) but have fundamental difference such e.g. senescence Cancer and aging are interconnected in both time and mechanisms and many of strategies can be used to target both, while in other cases antagonistic pleiotropy come in to effect and inhibition of one can be the activation of the other. 1.7 IS CANCER A TRANSMISSIBLE DISEASE? Two transmissible cancers referred to as DFT1 and DFT2 affects the Tasmanian devil. This cancer has led to a significant reduction of the devil population over the past 30 years with numbers decreasing by almost 70%. Canine transmissible venereal tumour (CTVT) is the oldest characterized transmissible cancer and affects dogs. CTVT, also known as Sticker’s sarcoma, is transmitted through direct contact including biting, licking, and sniffing tumour-affected, genital body parts. Dogs do not face extinction as the dog’s immune system plays a crucial role in spontaneous regression of CTVT. 9  Bivalves’ transmissible neoplasms (BTN) are transmitted via sea water. Molluscs lack a self/non-self-recognition system comparable to Major Histocompatibility Complex (MHC) found in vertebrates. The absence of MHC potentially increases their vulnerability to infectious malignancies. 1.7.1 In humans transmissibility is NOT a hallmark of cancer! The transmission of cancer between humans is very rare due to the immune system recognizing and rejecting foreign cells, thereby preventing the spread of cancer between individuals. However, specific circumstances allow such transmission of cancer, including organ transplantation and pregnancy. After organ transplantations, recipients have a suppressed immune system which facilitates the transmission of cancer from the donor. The compromised immune system fails to recognize and eliminate the cancer cells. The likelihood of receiving an organ from a donor with an undetected malignancy is extremely low (incidence rate of about 0.01%). Additionally, the genetic material are shared between the mother and the fetus during pregnancy. The placenta is permeable for cells, which allows the transmission of cancer cells from the mother to the fetus and vice versa. The transmission of cancer between fetus and mother is exceedingly rare. Furthermore, cancer transmission could also occur between twins, which was documented in some cases (twin to twin dissemination of leukaemia in utero). 1.8 CLASSIFICATION OF CANCER 1.8.1 Carcinoma Carcinoma are derived from epithelial cells Represent 90% of all cancers; largest class of cancer Tumours arising from epithelial cells forming protective layers are termed squamous cell carcinoma 10  Tumours arising from epithelia secreting substances into ducts or cavities (lumina) generate adenocarcinoma 1.8.2 Sarcoma Sarcoma derive from various connective tissues sharing a common origin in the embryonic mesoderm Constitute 1% of all tumours Sarcomas derive from a variety of mesenchymal cell types (fibroblasts, osteoblasts, adipocytes, myocytes etc) 1.8.3 Hematopoietic Tumours Hematopoietic malignancies are derived from blood-forming cells including immune cells and comprise about 8% of all human malignancies Leukaemia refers to malignancies of all hematopoietic cell lineages which are dispersed as single cell populations in the circulation Lymphomas include tumours of the lymphoid lineages (B, T lymphocytes) which aggregate to form solid tumour masses in lymph nodes Myeloma (plasmacytoma) is a cancer that forms in a type of white blood cell called plasma cell (producing antibodies). Myeloma localize in bone marrow/skeleton) o Acute lymphocytic leukaemia (ALL) o Acute myelogenous leukaemia (AML) o Chronic myelogenous leukaemia (CML) o Chronic lymphocytic leukaemia (CLL) o Multiple myeloma (MM) o Non-Hodgkin's lymphoma* (NHL) 11  o Hodgkin's lymphoma (HL) o *The non-Hodgkin's lymphoma types, also known as lymphocytic lymphomas, can be placed in as many as 15-20 distinct subcategories, dependina upon classification system. 1.8.4 Neuronal Tumours Neuroectodermal tumours derive from cells of the central and peripheral nervous system Around 1,5% of all diagnosed tumours Name of tumour Lineage of founding cell Glioblastoma multiforme highly progressed astrocytoma Astrocytoma astrocyte (type of glial cell) (Nonneuronal cell of central nervous system that supports neurons.) Meningioma arachnoidal cells of meninges (Membranous covering of brain.) Schwannoma Schwann cell around axons (Constructs insulating myelin sheath around axons in peripheral nervous system.) Retinoblastoma cone cell in retina (Photosensor for color vision during daylight.) Neuroblastoma (These tumours arise from cells of peripheral nervous system cells of the sympathetic nervous system.) Ependymoma glial cells lining ventricles of brain (Fluid- filled cavities in brain.) Oligodendroglioma oligodendrocyte covering axons (Similar to Schwann cells but in brain.) Medulloblastoma granular cells of cerebellum (Cells of the lower level of cerebellar cortex (as a related example)) 1.8.5 Other Cancers Other tumours which are atypical such as teratomas (rare disease) o Defy all attempts of classification o Teratomas arise from germ cell (egg and sperm) precursors that fail to migrate to proper destinations during embryonic development o Persist at ectopic (inappropriate) sites in the developing foetus o Teratomas show differentiated tissues found in a variety of adult tissues o Teratomas retain pluripotency of embryonic cells and represent the three layers of the embryo, i.e. endoderm, mesoderm and ectoderm o Teratomas develop recognizable structures of e.g. teeth, hairs, and bones o Teratomas progress to become highly malignant Melanoma derives from melanocytes (pigmented cells of skin and retina) o Melanocytes arise from primitive embryonic structures termed neural crest o Melanoma accounts for about 1,5% of cancer cases Anaplastic cancers o Small minority of cancers (2-4%) o Tissue-specific, differentiated traits of cells are lost 12  o Cells in anaplastic tumours are dedifferentiated o No histopathological criteria to identify tissue of origin o Anaplastic tumours are cancer of unknown primary (CUP) o Anaplastic tumours are of obscure origin. Histological appearance of an anaplastic tumour gives little indication of its tissue of origin. 1.8.6 TNM Classification of Cancer The TNM system is the most widely used cancer staging system for cancer reporting TNM is mostly used for carcinoma and sarcoma. Brain tumours and blood cancers use different system T refers to the size and extent of the main tumour. The main tumour is usually called the primary tumour N refers to cancer cell infiltration into nearby lymph nodes M refers to whether the cancer has metastasized into distant sites of the body Letters and numbers behind the TNM are for detailed description (e.g. T1N0MX or T3N1M0. T letters and numbers mean: o TX: Main tumour cannot be measured. o T0: Main tumour cannot be found. o T1, T2, T3, T4: Refers to the size and/or extent of the main tumour. The higher the number after the T, the larger the tumour or the more it has grown into nearby tissues N letters and numbers mean: o NX: Cancer in nearby lymph nodes cannot be measured. o N0: There is no cancer in nearby lymph nodes. o N1, N2, N3: Refers to the number and location of lymph nodes that contain cancer. The higher the number after the N, the more lymph nodes that contain cancer. M letters and numbers mean: o MX: Metastasis cannot be measured. o M0: Cancer has not spread to other parts of the body. o M1: Cancer has spread to other parts of the body 1.8.7 Grading of Cancer The grade reflects the aggressive potential of the tumour. “Low grade" (G1) cancers tend to be less aggressive than "high grade" cancers (G3) For example, breast cancer: Different "scoring systems" available for determining the grade. In scoring, three factors are taken into consideration: 13  1) Amount of gland formation (the cell “differentiation,” or how well tumour cells try to recreate normal glands) 2) Degree of nuclear "pleomorphism" or how "ugly" the tumour cells look 3) Mitotic activity, i.e. how many tumour cells are dividing 2 LECTURE 2: DNA DAMAGE AND GENOMIC STABILITY 2.1 CANCER DEVELOPMENT Cancer can basically develop from all cell types in the body Three stages of tumorigenesis: (1) tumour initiation, (ii) tumour promotion, and (iii) tumour progression Initiation: DNA damage and persistent mutations due to inefficient DNA repair or apoptosis causes uncontrolled cell proliferation Promotion: Initiated cells containing the mutations accumulate further mutations/epigenetic changes due to non-mutagenic (non-genotoxic) agents or events (e.g. inflammation) which stimulate uncontrolled proliferation Progression: further (epi)genetic changes occur leading to dissemination of cancer cells, i.e. metastasis (cancer cell invasion into surrounding etc) Vocabulary: o tumorigenesis o oncogenesis o carcinogenesis *terms are used synonymously for development of malignant tumour/cancer 14  Initiation and promotion reflect stages of tumour development which are reversible -> benign stages Progression indicates evolution of cells to an increasingly malignant stage (irreversible) Development of epidermal carcinomas in mice: Initiator: mutagenic (e.g. DMBA, a highly carcinogenic tar constituent) Promoter: non-genotoxic (TPA, a phorbol ester) 2.1.1 Terminology The word tumour and cancer include all abnormal types of cell growth Malignancy means that cancer cells invade the surrounding tissues as malignant cancer cells (see metastatic cascade) For epithelial cancers, carcinomas showing cancer cell invasion are malignant For epithelial cancers, adenoma are benign Neoplasia (neoplasms) includes benign and malignant growth of cells Carcinogenic means cancer-causing (can also be mutagenic but is not a must) 2.1.2 Time of Cancer Development Cancer requires 20 – 30 years to develop Red line: consumption of cigarettes Green line: mortality from lung cancer US population 2.1.3 Hallmarks of Cancer 1) Escaping the immune system: Cancer cells develop ways to evade detection and destruction by the immune system. 2) Escape from growth suppressors: Cancer cells lose responsiveness to signals that normally limit cell division. 3) Promoting proliferative signals: Cancer cells acquire mechanisms to sustain chronic proliferation. 4) Enabling replicative immortalization: Cancer cells activate pathways that allow them to divide indefinitely. 15  5) Invasion & metastasis: Cancer cells gain the ability to invade adjacent tissues and spread to distant sites in the body. 6) Evasion of cell death: Cancer cells resist programmed cell death (apoptosis). 7) Altered cellular metabolism: Cancer cells often exhibit changes in metabolic processes to support rapid growth. 8) Induction of angiogenesis: Cancer cells stimulate the formation of new blood vessels to supply nutrients and oxygen. 9) Genomic instability and mutations: Cancer cells accumulate genetic alterations that drive tumorigenesis. 10) Tumour-promoting inflammation: Cancer cells can exploit inflammatory processes to support tumour growth and progression. 2.1.4 Anatomical Protection of Cell Genomes Stem cells are less frequent targets of mutagenesis due to: anatomical shielding from toxins (by e.g. mucus secreted by crypt cells) Low mitotic activity Multiple drug resistance (pumping out of toxins) High DNA repair activity 2.1.5 Biochemical Protection of Cell Genomes Anatomical protection is not sufficient Efficient biochemical defence is required to protect the genome DNA is under constant attack by: Replication errors (DNA polymerase, microsatellite) Spontaneous changes in nucleotides (e.g. depurination) Mutagenic agents (chemical species, UV ray, X-ray) 2.1.5.1 Proofreading of DNA Polymerase Replication is powered by DNA polymerases -> pol-α, pol-δ, and pol-ε DNA pol-α, pol-δ, and pol-ε have proofreading activity to provide low level of errors or avoid errors Degradation of elongated strand with into 3‘->5‘ direction Loss of DNA proofreading causes endogenous cancer 16 2.1.5.2 DNA Repair - Mismatch Repair (MMR) Mismatch repair (MMR) monitors miscopied DNA sequences that were overlooked by proofreading of pols MMR critical in regions with repeated sequences such as mononucleotide repeats (e.g. AAAAAAA) or dinucleotide repeats (e.g. AGAGAGAG) DNA pol “stutters” while copying these repeats, resulting in longer or shorter versions of repeats in newly daughter strands Short repeats in the genome are termed microsatellites (thousands in the genome) Defective MMR leads to microsatellite instability (MIN) shrinkage or expansion of microsatellites Errors made: o 1/105 by DNA pol o 1/107 after proofreading of pol o 1/109 after MMR 2.1.5.3 Mutations by Endogenous Biochemical Processes Depurination of DNA -> chemical alteration -> spontaneous break of purine base (adenine or guanine) from deoxyribose (10.000 purine bases lost/day/cell) Depyrimidation of DNA -> 500 cytosines and thymine are lost/day/cell Deamination of DNA -> amine groups from cytosine, guanine and adenine are removed. E.g. deamination of cytosine leads to uracil which will be read as thymidine, causing a C-T point mutation (transition) Failure to repair (spontaneous) depurinated, depyriminiated or deaminated DNA cause point mutations 17 2.1.5.4 Reactive Oxygen Species Oxidation strongly affects damage of DNA Reactive oxygen species (ROS) generated by oxidation: O2 + e- -> O2- + e- -> H2O2 + e- -> OH- + e- -> H2O (superoxide ion, hydrogen peroxide, hydroxyl radical) ROS form covalent bonds with many molecules and attack bases within DNA and induce single- and double-stranded DNA breaks and DNA protein crosslinks Link to inflammation: Macrophages and neutrophils kill infected cells at inflammatory sites by phagocytosis using ROS production. In addition, collateral damage occurs by ROS acting as mutagens on genomes of nearby bystander cells -> chronic inflammation favours tumour development 2.1.5.5 Exogenous Mutagens X-rays (“ionizing radiation”): energy of X-rays is expended in stripping electrons from water causing ROS production -> creates single- and double-strand breaks (DSBs) of DNA. DSBs difficult to repair resulting in chromosome breaks Ultraviolet (UV) radiation: UV photons (UVA, UVB) generate pyrimidine dimers that is covalent bonds formed between two adjacent pyrimidines (T-T, C-C, or C-T dimers. Most are T-T dimers (60%). Pyrimidine dimers are very stable, but weak mutagenic. Pyrimidine dimers are efficiently removed only by nucleotide excision repair (NER) in human Physical shielding of keratinocyte nuclei from UV: cells protected from UV radiation using the melanin pigment transferred from melanocytes to keratinocytes Melanosome vesicles transfer melanin to keratinocytes in the basal layers of epidermis. Melanosomes assemble in supranuclear caps (sun umbrellas) above nuclei of keratinocytes (white arrows). Alkylating agents: chemicals that attach alkyl groups covalently to the DNA bases Alkylation of a DNA base destabilizes its covalent bond to deoxyribose resulting in the loss of purine or pyrimidine base from DNA or by misreading via the DNA pol machinery during replication. 2.1.5.6 Restoration of Normal Base Structure Reversal of DNA alkylation by DNA alkyltransferase which removes methyl and ethyl residues from the O6 position of guanine (O6 methylguanine-DNA-methyltransferase (MGMT) Absence of repair leads to G-A transition MGMT frequently silenced in tumours Loss of MGMT expression causes increased rates of mutations 18  The alkylating agent ENU (ethylnitrosourea) alkylates the O6 position of the guanine. MGMT restores the altered guanine to its normal structure. 2.1.5.7 Exogenous Mutagens Require Activation Large number of potent mutagens become altered by cellular metabolic processes (xenobiotic transformation). Xenobiotic transformation causes the conversion of “procarcinogens” to “ultimate carcinogen” An array of cytochrome P450 enzymes (CYPs) in the cell catalyse the transformations of carcinogens E.g. Benzo-pyrene (a molecule of the polycyclic aromatic hydrocarbons [PAH]) gets activate by CYP1A1 to an ultimate carcinogen -> Benzo-pyrene is a carcinogenic component of tobacco smoke and coal tar 2.1.6 Transformation of Xenobiotics Xenobiotics: chemicals that are foreign to the body, meaning they are not naturally produced by it. Examples include drugs, pollutants, and other substances that enter the body from the environment. Transformation of Xenobiotics refers to the series of biochemical reactions that convert lipophilic (fat-soluble) compounds into more hydrophilic (water-soluble) compounds, facilitating their excretion from the body. The two main pathways for the transformation of xenobiotics: 1. Detoxification: Phase 0: This is the initial phase where xenobiotics are imported into the cell. This can happen through active transport (requiring energy) or passive diffusion (no energy required, P-gp and Mdr). Phase I: In this phase, enzymes modify the xenobiotic molecule, often by adding or removing functional groups. This modification can make the xenobiotic more water- soluble, which aids in its elimination. Common modifications include oxidation, reduction, and hydrolysis. 19  Phase II: Here, enzymes conjugate the xenobiotic with another molecule, such as glucuronic acid, glutathione, or sulfate. This conjugation further increases water solubility and facilitates elimination. Phase III: The final phase involves the active transport of the modified xenobiotic out of the cell and into the bloodstream for excretion. This export is often mediated by proteins like P-glycoprotein (P-gp) and multiple drug resistance proteins (Mdr 1-6). 2. Toxification: In some cases, the metabolic transformation of a xenobiotic can lead to the formation of reactive intermediates. These intermediates can be highly reactive and can damage cellular components, leading to toxicity. Key Points: The overall goal of xenobiotic transformation is to make them more water-soluble and easier to eliminate from the body. The balance between detoxification and toxification is crucial. If detoxification pathways are overwhelmed or compromised, it can lead to toxicity. The efficiency of xenobiotic transformation can vary depending on factors such as the type of xenobiotic, individual genetic variations, and the overall health of the individual. Additional Notes: P-gp and Mdr (multiple drug resistance) proteins are important transporters that can both import and export xenobiotics, playing a role in both detoxification and toxification. Glutathione is a key antioxidant and plays a crucial role in detoxification by conjugating with xenobiotics. 2.1.6.1 Metabolism of Xenobiotics The metabolism of xenobiotics occurs in two phases: 1) Phase I: A chemical group is added to the xenobiotic -> activation of the compound occurs in phase I (but not exclusively) 2) Phase II: A second chemical group is added to the xenobiotic which allows the rapid transport for excretion and thus elimination The liver is the major site of the metabolism of xenobiotics! 2.1.6.2 Phase I Enzymes CYTOCHROME P450s: Contain heme which show in the presence of CO an absorption peak at 450 nm wavelength -> name P450 Is a superfamily with more than 500 members. Present in almost every cell type Metabolize endogenous and exogenous substances 20  Is the most important enzymatic system to metabolize xenobiotics Important CYP450: CYP1A1: polyaromatic hydrocarbon (not in the liver, lung, kidney, skin etc) CYP1A2: aromatic amines, Aflatoxin B1 (liver) CYP2E1: ethanol, drugs (liver) CYP3A4: Drugs, pre-carcinogens, Aflatoxin B1 (liver) 2.1.6.3 Phase II Enzymes Conjugation and binding to larger molecules Achieved by: Glucuronidation via UDP-glucuronosyl transferase (cofactor -> Uridindiphospho- glucuronic acid) Sulfatation via sulfotransferases (cofactor -> phosphoadenosinephosphosulfate) Methylation via methyl transferases (cofactor -> S-adenosylmethionine) Acetylation via N-Acetyl transferases (cofactor -> acetyl CoA) Conjugation of glutathione via glutathione-S-transferases (cofactor -> GSH) Cause the detoxification of cancerogenic substances! 2.1.6.4 Ultimate Carcinogens Form DNA Adducts Ultimate carcinogens attack the DNA and form covalent bonds with DNA bases resulting in DNA adducts DNA adduct formation is the reaction of a carcinogen with DNA DNA adducts activate NER and base excision repair (BER) The activated benzo-pyrene (BPDE) attacks the amine of guanine and forms the DNA adduct 2.1.6.5 Aflatoxin - Liver Carcinogen Aflatoxin B1 (mycotoxin from Aspergillus flavus; grow on grains) is metabolized by Cyp3A4 to the 8,9-epoxide which reacts with DNA to form DNA adducts. Aflatoxin 8,9-epoxide reacts with a guanine base pair, the most common form of DNA adduct formation leading to AFB1-N7-Guanyl. 21 2.1.6.6 Aflatoxin Causes p53 Mutations and Liver Cancer DNA adducts arising from Aflatoxin B1 cause mutations GC -> TA transversion is the most prevalent mutation Aflatoxin-DNA adducts cause “hotspot” mutation in p53 gene at which GC->TA transversion is prominent at the third position of codon 249, resulting in Arg->Ser missense mutation Ser249 mutation inactivates p53 and p53-mediated transcription leading to HCC p53 consists of 393 amino acids with functional domains and regions designated as mutational hotspots. Functional domains include the transactivation region (gold block), sequence-specific DNA-binding region (amino acids 100–293), nuclear localization sequence (dark green block) and oligomerization region (light green block). The majority of mutations are in sequence specific binding to DNA 2.1.6.7 Elimination of Abnormal DNA Nucleotides with abnormal chemical structures are not repaired by MMR, as MMR recognizes normal DNA structures Repair of DNA by mechanisms which recognize chemically altered bases in the DNA by base excision repair (BER) BER repairs lesions induced by ROS and depurination events BER recognizes chemically altered bases having minimal helix-distorting effect. DNA glycosylase cleaves bond between base and deoxyribose. Base-free deoxyribosyl phosphate is cleaved by AP endonuclease. Gap is filled by DNA pol-β and ligated. Nucleotide excision repair (NER) repairs DNA with chemically altered bases in the DNA and significant distortions of the DNA helix NER repairs lesion by pyrimidine dimers, DNA adducts. NER cleaves 24 nts on the 5’ side and 5 nts on the 3’ side, leading to a 29 nt single strand gap that is filled by DNA pol-δ or ε and ligated. 2.1.7 Error-Prone DNA Repair In some cases DNA replication occurs in still unrepaired DNA stretches, a process termed error-prone DNA replication Replication by error-prone DNA polymerases (also called bypass polymerases, e.g. DNA pol-β) DNA lesions are frequently not corrected leading to misincorporated bases (thus the term error-prone DNA repair is misleading) 22  Overexpression to by error-prone DNA polymerases in cancer cells enhance the mutation rate and tumour development DNA polymerase encounters the DNA lesion (thymidine dimer) and is most cases the error-prone DNA pol inserts the appropriate base but, in several percent, will instead incorporate a G-G dinucleotide 2.1.7.1 Familial Cancer Syndromes Due to Defects in DNA Repair 2.1.8 Xeroderma Pigmentosum (XP) XP patients have inherited defects in any of eight genes involved in NER XP patients show dry, parchment-like skin (xeroderma) and many freckles (pigmentosum) XP patients have 2.000-fold higher risk of skin cancer before age of 20 XP patients with defective NER have also higher risk for other disorder (e.g. neurological problems). Yet, UV (UVA, UVB) rays are the most important environmental mutagen for human and thus the most dangerous for XP. Strong sunlight can inflict 100.000 DNA lesions per skin cell per hour! Skin cancer appear with a median age of onset at about 60 yrs. In XP patients, skin cancer appears with a median age of 10 yrs. 2.1.9 Hereditary Non-Polyposis Colon Cancer (HNPCC) HNPCC is a familial cancer with predisposition to colon cancer (2-3% of all CRC). Less increased susceptibility to stomach, ovarian and urinary tract carcinoma. Adenoma-to-carcinoma progression occurs faster (2-3 yrs) instead of 8-10 yrs. HNPCCs have germ-line mutations in MMR genes (85% in MSH2 and MLH1; other 15% in MSH6 and PMS2). HNPCC patients inherit one defective allele. Arising tumour cells undergo loss of heterozygosity (LOH) and discard second wild type allele. High mutations in genes with microsatellite repeats. Type II TGF-β receptor (TGF-βRII) is mutant. Open reading frame contains a homopolymeric stretch of A10. Deletion from of A10 -> A8 causes nonsense mutation 23  and premature termination of translation. Strongly truncated TGF-βRII is not functional. Tumour cells escape growth inhibitory effects of TGF-β signalling. A variety of other genes with microsatellite repeats are affected by MMR deficiency (e.g. inactivation of apoptotic genes and other MMR genes. 2.1.10 Increased Cancer Susceptibility by other DNA Repair Defects BRCA1 and BRCA2 involved in maintenance of genomic integrity (considered as “caretakers”) Mutant germ line alleles of BRCA1 and BRCA2 confer susceptibility to breast and ovarian carcinoma 50% of familial breast carcinoma and 80% of familial ovarian carcinoma have mutant BRCA1/BRCA2 Carriers with mutant germ line alleles of BRCA1 and BRCA2 have a 50% risk of developing breast or ovarian carcinoma Patients inherit one defective allele. Arising tumour cells inactivate second wild type allele by promoter methylation (breast cancer) BRCA1 and BRCA2 are in a large complex with other proteins in nucleus, carrying RAD50 and RAD51 (both important proteins in repairing DNA breaks induced by X-ray) BRCA1 and BRCA2 localize at sites of double strand (ds) DNA breaks 2.1.10.1 Double strand DNA Breaks at Replication Forks Accidental dsDNA breaks at replication forks DNA is vulnerable to breakage at the single-stranded portions near the replication fork that are not replicated Breakage of single stranded region (termed “collapsed” replication fork) is equivalent to double strand break on already formed double helix 2.1.10.2 Homology-Directed Repair (HR) dsDNA breaks are repaired by HR Repair of dsDNA breaks in one chromatid by HR depends on the ability to include the undamaged, homologous DNA sequence in a sister chromatid (available after DNA replication) HR occurs during late S-phase or G2 phase Each of resulting ssDNA strands invades the undamaged sister chromatid which is unwound by the repair apparatus -> ssDNA strands from damaged chromatids are elongated 5’->3’ by DNA pol, using strands of sister chromatids as template Extended ssDNA are released and pair with another, allowing further elongation by DNA pol and ligation -> restoration of ds DNA helix 24 2.1.10.3 Interaction Partner of BRCA1/2 BRCA1 and BRCA2 act as scaffold to assemble DNA repair proteins BRCA2 recruits eight copies of RAD51 which bind coordinatively to the ssDNA strand BRCA1 binds the MRN complex composed of RAD50, Mre111 and Nbs1 which recognizes the end created by a dsDNA break and activated ATM 2.1.10.4 Nonhomologous End Joining (NHEJ) Limited degree of base pairing between ssDNA overhangs! HR is compromized in cells lacking BRCA1 and BRCA2 In the absence of BRCA1 and BRCA2, RAD51 is not properly recruited to the site of dsDNA break -> steps of HR do not occur correctly dsDNA breaks in cells with mutant BRCA1 and BRCA2 are repaired by NHEJ NHEJ is an error-prone procress since the alignment between the two DNA fragment being fused are not informed by wild type sequences of sister chromatids NHEJ occurs largely in G1 phase when sister chromatids are not available to allow HR 2.1.10.5 Therapy of BRCA Mutant Cancer Poly-ADP ribose polymerase (PARP)1 binds to ssDNA breaks and ADP ribosylates itself (addition of about 200 ADP ribose residues) ADP ribosylation of PARP1 attracts a series of DNA repair enzymes which get subsequently ADP ribosylated and then complete steps of BER In the presence PARP1 inhibitor, DNA repair enzymes are not recruited and BER cannot work The resulting ssDNA break converts to a dsDNA break after replication in S phase PARP1 inhibition works as therapy only in BRCA1/BRCA mutant cells, as otherwise the dsDNA break is repaired by HR (BRCA1/2 recruits RAD51) PARP1 inhibition used in clinics to treat ovarian and breast cancer 25 2.1.10.6 Update on PARP Inhibitors Used in Clinics (2020) FDA-approved PARP inhibitors 2.1.10.7 PARP Inhibitors and Mechanism of Action Mode of action of PARP inhibitors in chemo sensitization and radio sensitization. SSBs induced by chemical agents or ionizing radiation are repaired by PARP-dependent repair, resulting in cancer cell survival. Inhibition of PARP prevents DNA repair, resulting in cell death. The mechanisms of action of PARP inhibitors in synthetic lethality in HRdeficient cells. Endogenously induced DNA SSBs are normally repaired by PARP dependent SSB repair, resulting in cell survival. If PARP is inhibited, SSBs accumulate and cause replication fork collapse. This is usually repaired by HR, which involves BRCA1, BRCA2 and several other proteins preparing the DNA ends for the loading of RAD51 onto the ssDNA, enabling strand invasion into the complementary duplex as a template for DNA synthesis and high-fidelity DNA repair. In BRCA-mutant cells (deficient HR repair; HRD), fork collapse cannot be repaired, and the cell undergoes cell death. PARP inhibitors were initially thought to inhibit poly(ADP- ribosyl)ation and thereby cause cytotoxicity. However, the main cause of cancer cell death was found to be trapping of the PARP1 enzyme at DNA lesions by formation of DNA-protein crosslinks. ssDNA breaks caused by DNA damage are faithfully repaired in the presence of PARP1. However, trapped PARP1 enzymes can cause a threat to replication forks during the S phase leading to collapse of the replication fork. Fork collapse results in dsDNA breaks. In BRCA- proficient cells, homologous recombination (HR) enables the error-free repair of such breaks. By contrast, BRCA1/2- deficient cells are HR-deficient and rely on error-prone DNA end- joining pathways such as NHEJ to resolve the dsDNA breaks caused by fork collapse, triggering the accumulation of chromosomal aberrations and cell death by mitotic catastrophe. 2.1.10.8 Resistance to PARP Inhibitors Majority of patients inevitably develop resistance. Resistance occurs via one of three general mechanisms. 1. Alterations related to the drug as observed with chemotherapies, such as (A) upregulation of the efflux transporter P-glycoprotein, (B) downregulation of or mutations 26  in PARP1, which is restricted to cells expressing residual levels of BRCA1/2 or (C) loss of poly(ADP- ribose) glycohydrolase (PARG; enzyme which degrades poly ADP- ribosylation). 2. Restoration of homologous recombination (HR) can occur either through (D) reactivation of BRCA1/2 function or (E) loss of DNA end-protection, which is restricted to loss of BRCA1 and may occur via loss of the non-homologous end- joining (NHEJ) factor 53BP1. Loss of 53BP1 restores DNA end-resection in BRCA1-deficient cells (not in BRCA2- deficient cells) and rescues the HR defect and renders cells resistant to PARP inhibitors. 3. Restoration of replication fork stability via (F) increased protection from fork degradation, for example, (G) by loss of PTIP expression or loss of cell-cycle checkpoint arrest owing to loss of Schlafen 11 (SLFN11). Synthetic Lethality in Cancer Treatment Synthetic lethal interaction occurs between two genes. The perturbation (a mutation or inhibition) that affects either gene alone is viable but the perturbation of both genes simultaneously is lethal. PARP inhibitors act synthetic lethal with BRCA deficiency! The concept of synthetic lethality. (A) The loss or the inhibition of either of the protein products of gene A or B alone or the overexpression of gene A is viable. Mutation or (C) pharmacological inhibition of the protein product of gene B in cells with a mutation of gene A results in synthetic lethality. Overexpression of gene A and pharmacological inhibition of gene B results in synthetic lethality. Process is termed synthetic dosage lethality (SDL) 2.1.11 Risk of BRCA Mutant Carriers Carriers of a mutation in BRCA1 or BRCA2 have a risk of up to 80% of developing breast cancer and of 20% to 40% of developing ovarian cancer during the course of their life. Male mutation carriers generally do not become ill but can transmit the mutation to their progeny. Male carriers of a BRCA1 mutation have an increased risk of prostate cancer. Men with BRCA2 mutation have an increased risk of breast cancer. 2.1.12 Histon Function to Reduce Cancer Risk At sites of dsDNA breaks, H2AX (a variant of the histone 2A) gets bound and phosphorylated (by ATM and ATR kinases) resulting in γH2AX. γ-H2AX attracts DNA repair enzymes such as BRCA1 H2AX constitutes 15% of histones H2A and are involved in nucleosome formation. γ-H2AX flank the dsDNA break. 27 2.1.13 Chromosomal Alterations Cancer cells frequently show alterations of the karyotype Changes occur in the structures of individual chromosomes and in chromosome numbers Changes in the structures of individual unpaired chromosomes in solid and haematopoietic tumours Unrepaired dsDNA breaks (accidently occurring at DNA replication forks) are the major source of chromosomal translocations (e.g. bcr-abl). The Abl kinase (chromosome 9) is transferred to Bcr (chromosome 22) resulting in fused bcr-abl (in chronic myeloid leukaemia, CML). 2.1.13.1 Alterations in Chromosome Numbers Changes in chromosome numbers are observed in > 80% of carcinoma cells Changes in chromosome numbers are due to chromosomal instability (CIN) Changes in chromosome numbers associate with aneuploidy, i.e. the genome of the cancer cell contains extra copies of the individual chromosomes Changes in chromosome numbers are usually the consequence of missegregation of chromosomes during mitosis Sister chromatids are not accurately segregated during metaphase/anaphase of mitosis Checkpoint mechanisms fail to impose quality control on chromosomal segregation E.g. individual kinetochores at one sister chromatid become associated with too many spindle fibres which then pull into opposite directions -> results in stranded sister chromatid and aneuploidy Changes in chromosome numbers are the consequence of defects in the organization of spindle poles Cancer cells can generate multiple centrosomes in the interphase resulting in mitotic spindles with multiple poles The array of chromosomes will be divided among three or more daughter cells -> different numbers of chromosomes are segregated Segregation of chromosomes might lead to aneuploidy 2.1.13.2 Perturbation of Chromosomal Stability A variety of proteins are involved in chromosomal instability (CIN) E.g. Loss of retinoblastoma (Rb) causes deregulation of centrosome dublications, leading to aneuploidy Inactivation of Rb frequently is observed in cervical carcinoma cells which express the human papilloma virus (HPV) genome encoding viral E6 and E7 oncoproteins 2.1.14 Chemotherapy Alkylating agents generate DNA lesions difficult to repair Carboplatin works as alkylating agent but make additional crosslinks within strands Nucleoside analogues like gemcitabine interfere with nucleoside biosynthesis, and being incorporated into the DNA -> create residues that are difficult to replicate and/or repair Complex synthetic agents like etoposide inhibit topoisomerase 28  Natural products like paclitaxel stabilize/destabilize microtubules 2.1.14.1 Combination of Chemotherapeutics Emergence of drug resistance is inevitable Development of multidrug protocols to overcome drug resistance Combination of drugs with complementary modes of cancer cell killing such as e.g. a nucleoside analog plus an anti-metabolite plus a DNA crosslinker (FOLFOX) Probability of resistance to three drugs is very low -> high response rates of patients 3 LECTURE 5: TUMOUR EVOLUTION AND HETEROGENEITY 3.1 ONCOGENIC CELL TRANSFORMATION Focus formation was initially observed by infecting chicken embryo cells with Rous Sarcoma virus (RSV) carrying v-src Process of cell transformation, i.e. normal cells convert into tumour cells Transformation of normal (wild type, primary) cells with oncogene(s) results in the loss of contact inhibition (monolayer) and the formation of a multilayered clump of cells (a focus). 29  A single oncogenic point mutation (e.g. in Ras) cannot transform a normal cell into a tumour cell Myc plus Ras oncogenes transform rodent embryo fibroblasts Myc and Ras collaborate, i.e. Ras causes loss of contact inhibition and Myc causes immortalization More general: Ras-like oncogenes collaborate with Myc-like oncogenes in oncogenic cell transformation (mitogenic signals are responsible for triggered cells to go from C1 to S phase). Ras-like oncogenes: largely cytoplasmic oncoproteins involved in mitogenic signalling; Myc-like oncogenes: largely nuclear oncoproteins involved in deregulation of the cell cycle Transformation of rodent cells: two or more oncogenes are required 3.1.1 Oncogenic Transformation of Human Cells Oncogenic Ras plus Myc is insufficient for oncogenic transformation of normal human cells Telomere biology strongly differs between rodent and humans resulting in different responses to introduced oncogenes Immortalization of human cells is facilitated by adding the hTERT (telomerase) gene to other oncogenes (rodents have high TERT activity) Normal human (primary) cells require at least five or more genetic hits for oncogenic transformation (Ras, hTERT, inactivation of pRb, p53 and PP2A) In fact, four of these changes (activation of Ras and hTERT and inactivation of pRb and p53 are commonly observed in cells of human cancers (these represent driver mutations in some cancers, not all) Driver mutations confer growth advantage of cells while passenger mutations do not Oncogenic Cell Transformation can be induced through viral infection, not directly the virus induces malignancy, but the infection with oncoproteins DNA tumour viruses bearing multiple oncogenes are implicated in triggering human cancer, however, none of these viruses can fully transform normal human cells to tumour cells after infection 30  Additional genetic (mutations) or epigenetic (promoter methylation) changes are required to convert virus-infected cells to tumour cells This explains the disparities between the number of virus-infected people and the number of people with malignancies. E.g. about 90% of people in the West have Epstein-Barr virus (EBV) but only few develop Burkitt’s lymphoma Tumourigenic viruses: HBV, HCV, HPV, EBV, HTLV, SV40 Oncogenic transformation of human cells is not associated with cancer cell invasion and metastasis suggesting that the malignant transformation is partially incomplete Oncogenic cell transformation reflects the stage of tumour initiation 3.1.2 Monoclonal and Polyclonal Tumour Growth Tumours descend from a single ancestral cell (monoclonal development) Alternatively, tumours are composed of genetically distinct subpopulation of cells (polyclonal development) Proof that tumour population are monoclonal, however, is more complex Polyclonality (if occurs) could be easily abolished as one subpopulation of cells dominate in the overall subpopulation Monoclonal outgrowth becomes quite heterogenous due to the acquisition of new mutant alleles (genetic instability) -> this view complicates the assessment of the monoclonal origin of cancer Polyclonal tumours get monoclonal over time 3.1.3 Genetic Instability Population of cells within a tumour begin as a relatively homogenous collection of cells – constituting a monoclonal growth – but become quite heterogeneous due to acquisition of new mutant alleles  process termed genetic instability Genetic heterogeneity may mask the monoclonal origin of cancer cell population Nonetheless, vast majority of tumours are monoclonal growths descendant from single normal progenitor, termed cells-of-origin Precise identity of cell-of-origin often remains obscure 3.1.4 Mutator Phenotype Every cell in the tumour can be affected by mutation Some cancer cells drastically increase their mutation rate, i.e. some malignant cell carry genomes that are far more mutable than genomes of normal cells – a condition termed mutator phenotype Increase in mutation rate due to mutations in genes belonging to the DNA repair machinery and genetic stability Repetitive cycles of mutagenesis and selection mimic Darwinian evolution mutations in the DNA repair system = > inefficient 31 3.2 TUMOUR DEVELOPMENT By the time a solid tumour is detected, it frequently measures 1 cm3 and encompasses 108–109 cells, each cell containing thousands of clonal, subclonal and random mutations Passenger mutations do not equal driver mutations For cancers that require only one or two mutations, such as inherited retinoblastoma, a mutator phenotype may not be necessary (retinoblastoma needs a few mutations, some cancers need more mutations but there is a limit of mutations since if there are too many, the cells die due to a mitotic catastrophe) However, for most cancers that require three or more driver mutations, a mutator phenotype may be inevitable Probably a maximum mutation frequency that a tumour can tolerate: a further increase would be detrimental, reducing cell fitness and enhancing cell killing (as induced e.g. by chemotherapy) 3.2.1 Knudson Model – Two-Hit Hypothesis Both alleles of a tumour suppressor (TS) gene must be inactivated by “null” mutations (e.g. nonsense mutations or deletions) or epigenetic silencing to develop tumour (two- hits hypothesis) In inherited retinoblastoma (tumour of retina), first inactivation of Rb allele is inherited and second inactivation of Rb allele is acquired -> early onset of retinoblastoma In non-inherited retinoblastoma, first and second inactivation of Rb alleles are acquired -> later onset of retinoblastoma Are two null mutations of tumour suppressor alleles essentially required for tumorigenesis? NO! 3.2.2 The Role of p53 as a TS Cell-physiological stresses can induce p53 levels p53 protein causes a cytostatic response (cell cycle arrest, either irreversible (“senescence”) or reversible (“return to proliferation”). DNA repair proteins may be mobilized as well as proteins that alter metabolism or block of angiogenesis. p53 can also trigger apoptosis p53 levels are controlled by various kinases. Extensive ssDNA regions and double-strand DNA breaks (DSBs) activate two kinases, ATR and ATM, respectively. These kinases phosphorylate p53 directly, or indirectly via Chk1 and Chk2, at its N- terminal domain. This phosphorylation prevents the binding of the p53 antagonist Mdm2, thus stabilizes p53 protein levels 3.2.3 Dominant Function of Mutated TS Alleles p53 is a tumour suppressor (TS) which is mostly affected by missense mutations resulting in amino acid substitutions Mutated p53 allele interferes/abolishes the activity of the p53 wildtype allele contributing to malignant cell transformation Such alleles are termed “dominant-interfering” or “dominant negative” alleles p53 acts as a tetrameric transcription factor (TF). The mutant allele (subunit) generates tetramers resulting in inactive p53 because of defective DNA binding 32  Heterozygosity of mutant p53 results in >90% of p53 tetramers (fifteen-sixteenths) lacking normal TF function p53 functions as a dominant-negative allele Knock-in of a point mutation in the DNA-binding domain of p53 into one p53 gene copy causes loss of almost all p53 function In contrast, when one p53 gene copy was completely inactivated (yielding a null allele), p53 function was almost normal 3.2.4 Hyperplasia (Epithelium) Development of tumours is a complex, multistep process Cells at the very early phase deviate only minimally from normal tissues and show excessive number of cells, termed hyperplastic growth Hyperplastic tissues appear reasonably normal 3.2.5 Dysplasia Slightly more abnormal tissue is dysplastic Dysplasia show abnormal cytological changes Abnormal cytological changes include variability in nuclear size and shape, increased ratio of nuclear versus cytoplasmic size, increased mitotic activity Dysplasia associates with changes in overall tissue architecture Dysplasia is a transitional state between complete benign and premalignant state Cells have not broken through the basement membrane (white dashed line) and show increased mitotic activity (white arrows) 33 3.2.6 Adenoma More abnormal tissue is termed adenoma (epithelial tissue) Adenomas are large growths which can be detected by eye (e.g. adenomatous polyps in colon, papillomas in skin) Adenoma do not penetrate the basement membrane which separate neoplastic cells from the tumour-stroma Adenomas are considered as benign Adenomas develop to carcinoma in situ (still not broken through the basement membrane) which are malignant because carcinoma develop to invasiveness Polyp of the colon (left) and ductal carcinoma in situ (right; DCIS; breast carcinoma). DCIS has not invaded through the basement membrane 3.2.7 Tumour Development of Epithelial Tissues Multi-step progression showing hyperplastic, dysplastic and adenomatous growth to carcinoma is best documented in colon due to colonoscopy, but other tissues exhibit similar growth patterns Multi-step tumour progression more fragmentary in non-epithelial tissues (nervous system, connective tissue, hematopoietic system) 3.2.8 Colon Carcinoma Loss of APC (adenomatous polyposis coli) by loss of heterozygosity (LOH Chr. 5q) Activation of K-Ras by mutation Loss of p53 by LOH Chr. 17p Loss of Smad4 and other tumour suppressor genes (TSGs) by LOH Chr. 18q Widespread hypomethylation by loss of methylated CpGs (probable activation of transposable elements wreaks genetic havoc) Loss of APC is always first (like in FAP -> APC+/- ) but order of subsequent changes vary from to tumour to tumour 80% of patients APC inactivation; 35% K-Ras mutation; < 50% loss of p53; 60% loss of Smad4 and other TSGs 3.2.9 Field Cancerization Sporadic tumours sprout multiple, apparently independently arising tumours, a phenomenon termed field cancerization. Two or more premalignant growths erupt suddenly, separated by apparently normal epithelium. 34  Initiating mutation in clonal descendants acquire a second mutation and form a patch of cells. Cells in the patch acquire independent mutation and become histologically abnormal neoplastic cells Tumours of field cancerization arise independently although they derive from same clone of initiated cell 3.2.10 Darwinian Evolution Darwinian view: Evolving units are individual cells competing with one another in a population of cells Mutations create genetic variability and forces of selection favor outgrowth of individual cells and their descendants with advantageous traits (proliferation, survival etc) leading to clonal expansion Darwinian model is simplistic (e.g. not considering type of mutation or cancer stem cells) 3.2.10.1 Driver and Passenger Mutations Mutations are random and rarely hit critical genes which when mutated confer advantageous phenotypes driving clonal expansion Advantageous genetic changes are driver mutations and cause clonal expansion Mutations irrelevant for tumour development are termed passenger mutations Driver mutations are biologically important for tumour development and cancer therapy Only low proportions of mutations (< 5%) are driver mutations 3.2.11 Parallel Clonal Expansion Genomes of cells become increasingly unstable during cancer progression Rate at which new mutants are generated may exceed the rate at which less phenotypically less fit clones are eliminated Tumours develop an increasing number of sectors dominated by genetically distinct subclones resulting in a process of dynamic clonal diversification 3.2.12 Darwinian Model The Darwinian model is simplistic and remains a rather theoretical construct Outlines of the model are true but difficult to validate What are the key genetic changes responsible for clonal expansion (driver mutations)? What are the key epigenetic changes (e.g. promoter methylation) responsible for clonal expansion? What is the kinetics of each step of multi-step progression – how long does each step take? E.g. rate of oncogenic point mutations (e.g. Ras) occur with a frequency of 10 -6 to 10-7 per cell generation, while frequency of LOH is 100-fold higher. Frequency of promoter methylation is 10-4 per cell generation Impact of the mutator phenotype? Impact of cancer cell plasticity by e.g. dedifferentiation (EMT) and its reversal? Impact of cancer cell competition but also the synergistic collaboration of cancer cells? Accumulation of these processes results in intra-tumoral heterogeneity (within individual tumour mass) and inter-tumoral heterogeneity (within individual cancer patients) 35 3.2.13 Cancer Stem Cells Not all cells of the (pre)-neoplastic clone are equivalent E.g. Minor population of CD44high/CD24low breast carcinoma cells generate new tumour in mice while majority cell population fail to do Tumour-initiating cells, i.e. cancer stem cells (CSCs) demonstrated in leukaemia and solid tumours such as breast, pancreatic, liver, colorectal and brain tumours Stems cells/CSCs divide asymmetrically to transit amplifying cells/differentiated cells and divide periodically (not continually) to produce large numbers of descendants Left: Canonical hardwired stem cell/CSC hierarchy. Stem cells/CSCs are rare and relatively quiescent. Upon asymmetric division, they give rise to one stem cell and one transient amplifying (TA) cell. The latter divides rapidly, yet it is not capable of self- renewal and eventually undergoes differentiation. Non-stem cells are poorly tumorigenic and display limited plasticity. Right: Novel features of stem cell/CSCs hierarchies. Stem cells/ CSCs are not necessarily rare or quiescent and are instructed by niche signals following neutral competition dynamics. TA cells and differentiated cells can be reprogrammed into stem cells by the niche through plasticity. Tumour-initiating cells/CSCs show low proliferation, high chemoresistance and high DNA repair activity Tumour-initiating cells/CSCs are resistant to conventional chemotherapy and radiotherapy Consequences of anti-CSCs therapies: In tumours displaying a unidirectional hardwired CSC hierarchy, elimination of CSCs is sufficient to cure the disease In the case of CSCs having extensive cell plasticity, niche signals will reinstruct stem cell properties to progenitor or differentiated cells after CSC loss, which will result in tumour regeneration and therapy failure Blocking niche signals will be more effective for this class of tumours and may improve therapeutic efficacy by preventing plasticity and CSC regeneration 3.3 PROMOTION Development of epidermal carcinomas in mice: Initiator: mutagenic (DMBA) Promoter: non-genotoxic (TPA) Initiated cells containing (driver) mutations accumulate further mutations/epigenetic changes due to nonmutagenic (non-genotoxic) agents or events (e.g. inflammation) Clonal expansion of cancer cells 36 3.3.1 Tumour Promotion by Non-Genotoxic Agents Striking example of tumour promotion is provided by head-and-neck cancers caused by tobacco and ethanol -> cigarette smoking in combination with consumption of distilled alcohol leads to a 100-fold increased risk Cigarette smoke/tobacco tar is rich in mutagenic carcinogens (initiating agent) -> regeneration of epithelial cells Ethanol is a weak mutagen (non-genotoxic) but has toxic effects on epithelial cells lining the mouth and throat (promoting agent) -> induces necrosis High percentage of ethanol (hard drinks) induces cell death of epithelial cells. Stem cells carrying a mutation induced by tobacco tar start uncontrolled proliferation in order to regenerate the tissue -> clonal expansion 3.3.2 Tumour Promotion by Mitogenic Agents Prominent examples are steroid hormones (oestrogen, progesterone and testosterone) in female for programming the proliferation of cells in reproductive tissues Monthly menstrual cycle between menarche and menopause results in proliferation and regression of epithelia of mammary gland and endometrial lining of the uterus Lifetime breast cancer risk decreases by delayed menarche and early onset of menopause Removal of ovaries (prime source of oestrogen) reduces breast cancer risk to plummet Effects of oestrogen are quite complex but may stimulate clonal outgrowth of initiated mammary epithelial cells 3.3.3 Tumour Promotion by Inflammation Few tumour promoters are cytotoxic. Great majority is chronic inflammation Prominent examples of chronic inflammation driving clonal expansion if cancer cells o hepatocellular cancer by HBV/HCV-induced hepatitis o hepatocellular cancer by non-alcohol-induced steatohepatitis (NASH) -> obesity o gastric cancer by Helicobacter pylori-induced gastritis -> infectious disease o oesophageal carcinoma by chronic acid reflux o mesothelioma by asbestos -> inducer of inflammation o colorectal carcinoma by inflammatory bowel disease o lung carcinoma by chronic bronchitis o gallbladder carcinoma by chronic cholecystitis o pancreas carcinoma by pancreatitis 3.3.3.1 Inflammation in Infection vs Inflammation in Cancer Damage to epithelial tissues caused by injury or infection causes activation of myeloid cells which start to produce inflammatory cytokines to activate innate and adaptive sterilizing immunity to get rid of the pathogen and to activate epithelial cell proliferation to close down the barrier dysfunction which allowed translocation of pathogen or to repair inflicted injury. After this concerted effort insulted epithelial tissue comes back to normal state of homeostasis. 37  If initial disturbance of epithelial homeostasis is caused by an oncogenic event, the sterilizing immunity will not remove the initial insult and the enhanced inflammation and cytokine-driven proliferation will facilitate tumour growth rather than restoring normal epithelial homeostasis. 3.3.3.2 Cancer-Related Inflammation Molecular basis of cancer-related inflammation: Inflammation or oncogene activation results in the expression of pro-inflammatory transcription factors within tumour cells (such as NF-κB, STAT3 or HIF1α) These activated transcription factors mediate expression of cytokines and chemokines (including TNFα and IL-6) as well as inflammatory enzymes (such as COX-2), forming inflammatory responses within the tumour microenvironment Host leukocytes including macrophages, dendritic cells, mast cells and T cells are recruited by chemokines within the tumour stroma to mediate the immune response Inflammatory enzymes catalyse key steps in prostaglandin synthesis, which further regulate processes involved in cancer related immunity and inflammation COX-2, cyclo-oxygenase-2; HIF1α, hypoxia-inducible factor 1α; IL-6, interleukin-6; NF-κB, nuclear factor-κB; STAT3, signal transducer and activator of transcription 3; TNFα, tumour necrosis factor-α. 450 million people worldwide chronically infected with HBV; 200 million people with HCV HBV and HCV unrelated to another (genome, replication) but act similarly in hepatitis HBV/HCV cause proliferation of hepatocytes (normally do not divide) as hepatocytes killed by HBV/HCV are replaced (regeneration) 38  HBV/HCV infections cause infiltration of immune cells 3.3.3.3 Promotion of Liver Cancer by NASH Prevalence of non-alcoholic fatty liver disease (NAFLD) is 24% of general population which can develop to non-alcoholic steatohepatitis (NASH; 350 million NASH patients worldwide) Hepatic substrate overload (high fructose, high cholesterol, fat-tissue-derived free fatty acids etc) causes NAFLD/NASH development. The augmentation of intrahepatic CD8+ T cells, T helper 17 (TH17) cells, natural killer T (NKT) cells and infiltrating inflammatory macrophages along with inflammatory cytokines, can lead to chronic necro- inflammation, facilitating NAFLD to NASH transition. These disease states are to some degree reversible. -> steatosis develops to NASH due to lipotoxic stress Chronic hepatocyte cell death and compensatory proliferation during NASH with mild to advanced fibrosis (activation of hepatic stellate cells; HSCs), together with increased levels of TNF superfamily (TNFSF) members, TGF-β and IL-17, contribute to increased hepatocellular carcinoma (HCC) risk. ER, endoplasmic reticulum; PRR, pattern recognition receptor. Metabolic stress induced by a chronic hypercaloric, high-fat or high-fructose diet causes a metabolic disturbance in hepatocytes, leading to increased ROS, ER stress 39  and oxidative stress in hepatocytes, resulting in apoptosis and necroptosis of hepatocytes, which induce an inflammatory hepatic reaction. Incoming adaptive and innate immune cells affect hepatocyte metabolism through cytokine expression and induce a metabolic reprogramming of hepatocytes, characterized by downregulation of hepatic genes involved in lipolysis and β-oxidation. Moreover, these metabolic reprogramming leads - together with chronic hepatocyte damage - to enhanced cell death, DNA damage, compensatory hepatocyte proliferation and further increased immune cell activation, which activates HSCs and fibrosis that drive pro-carcinogenic processes. 3.3.4 Tumor Promotion – NF-kB Signalling NF-kB signalling links inflammation to tumour promotion NF-kB induces anti-apoptotic genes (e.g. BCL-XL) and inhibits the apoptosis of epithelial cells NF-kB induces the proliferation of epithelial cells by upregulation of e.g. cyclin D1 or c- myc NF-kB induces cyclooxygenase-2 (COX-2) which is responsible for the production of prostaglandin E2 (a pro-inflammatory molecule) in epithelial cells NSAIDS, non-steroidal anti-inflammatory drugs such as aspirin block inflammation and tumour progression NF-kB induces the cytokine TNF-α, a cytokine attracting immune cells into the stroma Blocking TNF-α does not avoid tumour initiation (dysplasia) but strongly impairs tumour promotion (transition from dysplasia to carcinoma) 3.3.4.1 Chronic inflammatory crosstalk between cancer cells and immune cells NF-kB in immune cells is initially activated by TNF, IL-1 and various pathogen- associated or damage-associated molecular patterns, leading to the production of proinflammatory cytokines, chemokines and growth factors such as TNF, IL-1, IL-6 and VEGF Pro-inflammatory cytokines activate NF-kB and STAT3 in cancer cells and in components of the tumour microenvironment, resulting in the stimulation of cancer cell proliferation and survival, EMT, angiogenesis and metastasis Cancer cells recruit more immune cells into the tumour microenvironment by producing chemokines and thereby augment and maintain the local inflammatory state, thus establishing a chronic feedforward loop that enhances tumorigenesis 3.3.5 Cancer Prevention by Anti-Inflammation Anti-inflammatory agents in cancer prevention Aspirin (NSAID) is cancer-preventive agent. Since 2015, the US Preventive Services Task Force has recommended the routine use of aspirin for CRC prevention among individuals aged 50–59 years who have a high risk of cardiovascular disease and a low risk of bleeding. 3.3.6 Tumour Development by Altered Metabolism Cancer cells exhibit altered metabolism from early to late stages of tumour development One of these important metabolic shifts of cancer cells is aerobic glycolysis (production of lactate from glucose) 40  Aerobic glycolysis is not an invention of cancer cells. Highly proliferating normal cells exhibit aerobic glycolysis as well Are metabolic shifts causes or consequences of the growth state of cancer cells? Breakdown of glucose in normal cells under oxygen conditions (normoxia) o In most normal non-proliferating cells having access to adequate oxygen, glucose is imported into the cells by glucose transporters (GLUTs) and then broken down by glycolysis and the citric acid cycle. During the last step of glycolysis, pyruvate kinase form M1 (PKM1) ensures that its product, pyruvate, is imported into the mitochondria, where it is oxidized by pyruvate dehydrogenase (PDH) into acetyl CoA for processing in the citric acid cycle. Altogether, the mitochondria can generate as much as 36 ATP molecules per glucose molecule. o Enzymes are in rectangles, glucose metabolites are in ovals, low–molecular- weight compounds are in hexagons, regulatory proteins are in pentagons. Breakdown of glucose in cancer cells (aerobic glycolysis or Warburg effect) o In cancer cells, including those with access to ample oxygen, the GLUT glucose transporters import large amounts of glucose into the cytosol, where it is processed by glycolysis. However, as the last step of glycolysis, pyruvate kinase M2 (PK-M2) causes its pyruvate product to be diverted to lactate dehydrogenase (LDHA), yielding the lactate that is secreted in abundance by cancer cells. Because relatively little of the initially imported glucose is metabolized by the mitochondria, as few as 2 ATP molecules are generated per glucose molecule. Moreover, many of the intermediates generated during glycolysis are diverted toward biosynthetic uses. This mode resembles the metabolic state of normal, rapidly dividing cells, which also divert a significant portion of their glycolytic intermediates to biosynthetic pathways. o Enzymes are in rectangles, glucose metabolites are in ovals, low–molecular- weight compounds are in hexagons, regulatory proteins are in pentagons. o Aerobic glycolysis is not an invention of cancer cells. Highly proliferating normal cells exhibit aerobic glycolysis as well. Facts associated with aerobic glycolysis o PK-M1 is commonly expressed, but PK-M2 is highly expressed in cancer cells. PK-M2 diverts pyruvate to cytosol where it is metabolized to lactate by LDH-A o PK-M2 has a slow turnover number which favours the formation of lactate rather than pyruvate o PK-M2 physically interacts with proteins containing phosphotyrosine residues causing a shutdown of its catalytic activity o Reduced PK-M2 activity results in the accumulation of glycolytic intermediates which are important for the biosynthesis of nucleotides and lipids (cell division!) o GLUT1 and LDH-A highly upregulated in cancer cells Cancer cells use aerobic glycolysis and o overcome limited access to oxygen (hypoxia) and to adapt to this oxygen starvation o import enormous amounts of glucose from the surrounding by elevated levels of glucose transporters, particularly GLUT1. Expression of GLUT1 is driven by oncogenes including myc, ras and src as well as hypoxia (HIF-1α) -> large growth advantage by high GLUT1 expression in vivo 41  o use only 1% of glucose for ATP production while 30% of glucose is used for ATP production in normal cells 3.4 CANCER PROGRESSION AND METASTASIS Cancer progression is linked to the dissemination (spreading) of cancer cells from the primary site to distal organs Cancer progression is considered as an irreversible, malignant stage Metastasis occurs in sequential steps Linear progression: clonal selection; advantageous clones expand and dominate over the others with additional mutational changes, resulting in tumour heterogeneity. Heterogeneous cells colonize different organs. Metastatic colonies carry mutations/changes as in the primary tumour plus additional ones. Parallel progression: Additional site-specific subclonal changes occur that endow disseminating tumour cells additional metastatic properties that are needed for the formation of overt metastases. Metastatic colonies carry mutations/changes different from the primary tumour. Progression by „try and error“ or search for better conditions due to hostile conditions (e.g. hypoxic conditions) 42 3.4.1 Lymphatic and Haematogenous Spread Invasion through the extracellular matrix (ECM) towards vessels. Cells intravasate into either a blood or lymphatic vessels. Cells undergoing lymphatic spread transit to a lymph node, where they become passively trapped, and can grow to form a lymph node metastasis. Cancer cells from lymph node metastases can further disseminate out of the lymph node through efferent lymphatic or blood vessels, entering circulation. Cancer cells undergoing haematogenous spread must persist through both the physical stresses of blood circulation and apoptosis caused by lack of attachment to the ECM in order to survive as circulating tumour cells (CTCs). CTCs arrest at a distant site due to either passive trapping in a vessel too narrow for them to pass through or active adhesion to the endothelial cell layer. After extravasating out of circulation, these disseminated tumour cells (DTCs) must survive in the new organ environment to seed micrometastases. After escaping any state of dormancy induced by the environment, these micrometastatic seeds can undergo outgrowth into a macrometastasis 3.4.2 Organotropism of Disseminating Tumour Cells (DTCs) Organ-specific pattern of metastasis (organotropism) Tumour cell (“SEED“) as well as the colonized organ („SOIL“) constitute organ specificity of metastasis. Organotropisms depend on mechanical factors (e.g. blood flow) and molecular interactions (e.g. receptor/soil and ligand/seed). Major sites of metastasis: o lung o bone o brain o liver o body cavities (peritoneum, pleura) Dissemination of cancer cells via blood, lymph or cavities! 43 3.4.3 Metastasis is an Inefficient Process Dissemination of primary cancer cells via blood, lymph or cavities Tumour with 1 cm3 containing 1 billion (109) cells show dissemination of 0.01% of cells After intravasation, less than 0.01% of cells develop metastatic lesions Human body contains about 35 trillion (3.5 x 1013) cells Lifetime of cells: white blood cells 14 days, red blood cells 120 days, liver cells up to 18 months, brain cells/neurons stay throughout life Lifetime of metastatic cells? 3.5 CANCER CELL DORMANCY 3.5.1 Tumour Dormancy Tumour dormancy is a crucial trait allowing disseminating tumour cells (DTCs) to survive, adapt and colonize at distant organ in the interval of latent malignant progression (latency) -> definition of tumour dormancy Differences in dormant mechanisms: Mechanisms of dormancy in the primary tumour (e.g. drug-tolerant persister cancer cells) are considered to be different from dormancy of metastatic cells Short latency: malignant cells in the primary tumour acquired most of metastatic traits to overtake organs immediately after infiltration. Long latency: circulatory tumours cells (CTCs) in blood and disseminating tumours cells (DTCs) in tissue need time to acquire functions for colonization at secondary sites 3.5.2 Temporal Course of Metastasis Different length of latency: o short (lung cancer) o middle (colon cancer, ER- breast cancer) o long (prostate cancer, ER+ breast cancer, but also melanoma and renal cancer) Temporal Course of Breast Cancer Metastasis o Time to relapse (recurrence): ▪ ER- breast cancer within 5 years ▪ ER+ breast cancer up to decades 44 3.5.3 Dormancy and Minimal Residual Disease Dormant tumour cells are linked to o Cell cycle arrest o Genetic alterations o Stemness o Evasion of immune response o Interaction with the microenvironment Minimal Residual Disease o Undetectable dormant metastatic cells that survived treatment contribute to minimal residual disease o Metastatic cells from minimal residual disease can be transferred through organ transplants o E.g. organs from melanoma patients successfully treated and clinically disease- free for over 10 years develop metastasis after transplantation 3.5.4 Mechanisms of Tumour Dormancy Cellular dormancy: a single DTC can undergo growth arrest, also called solitary cell dormancy Tumour mass dormancy: expansion of micrometastatic lesions (< 1 mm) showing similar rates of proliferation and apoptosis; growth arrest can also occur 3.5.5 Niches of Metastasis (Bone Marrow) Niches promote cell survival and cause exit from dormancy o Perivascular niche: neovascularization induce micrometastatic outgrowth by TGF-β o HSC niche: protective by secretion of CXCL12 o Osteogenic niche: heterophilic cadherin interaction enhance mTOR and outgrowth 45 3.5.5.1 Osteolytic Bone Metastasis 3.5.6 Models to Study Tumour Dormancy in vitro and in vivo Limitations to study dormancy in vivo: Tracking asynchronous disseminated metastatic cells difficult Single cell resolution often impossible Removal of single stromal cells difficult Limitations to study dormancy in vitro: Do not reflect the complexity (e.g. tissue architecture, microenvironment/stromal cells, oxygen levels etc) 46 3.5.7 Therapeutic Strategies Targeting Tumor Dormancy 3.5.8 Key Question: Why metastasis happens? Possible answers: o Hostile conditions (oxygen/nutrients) o Organ formation at the wrong site at the wrong time (activation of developmental mechanisms) o Attack by immune cells