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Cancer biology Dr. med. vet. Matti Kiupel, MS, PhD, DACVP Associate Professor, Section Chief Anatomic Pathology Michigan State University Diagnostic Center for Population and Animal Health 4125 Beaumont Road, Room 152A Lansing, MI 48910, USA Tel.: ** 517 432 2670 E-mail: [email protected] WHAT IS CANCER? Cancer is a group of diseases characterized by the uncontrolled growth and spread of abnormal cells. Cancer Facts & Figures - 2005 American Cancer Society www.cancer.org WHAT IS ONCOLOGY? Greek oncos = tumor The study of neoplasms or neoplastic cells. The Genetic Basis of Human Cancer, Second Edition B. Vogelstein and K.W. Kinzler (Editors) McGraw-Hill, New York, 2002 1 WHAT ARE NEOPLASMS? NEOPLASIA: “NEW GROWTH” Growth exceeds that of the normal tissues Growth is uncoordinated Persists in the same excessive manner after cessation of the stimuli which evoked the change Robbins Basic Pathology, 7th Edition V. Kumar, R.S. Cotran, and S.L. Robbins (Eds) Saunders, Philadelphia, 2003 WHAT ARE TUMORS? TUMOR: “A SWELLING” Any "lump" or 3-dimensional swelling Described in the 1st century A.D. by Celsus as one of the 4 cardinal signs of inflammation: Rubor, tumor, calor, and dolor; functio laesa (Virchow) WHAT ARE TUMORS? Result of a disease process in which a single cell acquires the ability to proliferate abnormally, resulting in an accumulation of progeny cells. The Genetic Basis of Human Cancer, Second Edition B. Vogelstein and K.W. Kinzler (Editors) McGraw-Hill, New York, 2002 2 WHAT IS CANCER? Cancers are those tumors that have acquired the ability to invade the surrounding normal tissues. The Genetic Basis of Human Cancer, Second Edition B. Vogelstein and K.W. Kinzler (Editors) McGraw-Hill, New York, 2002 CANCER SIGN OF THE ZODIAC The Crab Cancer in Mythology The Second Labor of Hercules The Lernaian Hydra 3 WHY IS CANCER DEPICTED AS A CRAB? Hippocrates of Cos, 460-375 BC coined from the Greek word meaning crab the terms karkinos (for benign) and karkinõma (for malignant) to describe solid tumors. Neoplasia. In: Anderson’s Pathology, Volume 1 G.T. Diamandopoulos and W.A. Meissner (1985) J.M. Kissane (Editor), C.V. Mosby, St. Louis WHY IS CANCER DEPICTED AS A CRAB? Galen, AD 131-201 “As a crab is furnished with claws on both sides of the body, so in this disease the veins which extend from the tumor represent it a figure much like that of a crab” In: Cancer, Volume 1 W.R. Bett (1957) Historical aspects of cancer. R.W. Raven (Editor), Butterworth, London WHY IS CANCER DEPICTED AS A CRAB? Paul of Aegina, AD 625-690 “…some say that cancer is so called because it adheres with such obstinacy to the part it seizes that, like the crab, it cannot be separated from it without great difficulty...” In: Cancer, Volume 1 W.R. Bett (1957) Historical aspects of cancer. R.W. Raven (Editor), Butterworth, London 4 WHAT IS CANCER? “Malignant tumors are collectively referred to as cancers, derived from the Latin word for crab – they adhere to any part that they seize on in an obstinate manner, similar to a crab.” Robbins Basic Pathology, 7th Edition V. Kumar, R.S. Cotran, and S.L. Robbins (Eds) Saunders, Philadelphia, 2003 TUMORS AND CANCER Different Names for the Same Pathologic Lesion? Neoplasm, Tumor, and Cancer Do all of these terms refer to the same pathological lesions? Not all neoplasms are cancerous... However, all cancers are considered to be “tumors” CLASSIFICATION OF NEOPLASMS Hematopoietic Leukemias Solid tumors Sporadic or Hereditary 5 CLASSIFICATION OF NEOPLASMS Benign Tumors Malignant Tumors Not all neoplasms are cancerous! CLASSIFICATION OF NEOPLASMS In oncology, the division of neoplasms into benign and malignant categories is extremely important This classification is based on a judgement of a neoplasm’s potential clinical behavior Malignant tumors are collectively known as cancers CLASSIFICATION OF NEOPLASMS What characteristics of a neoplasm suggest that it is benign versus malignant? Cellular Features Tumor Growth Pattern Clinical Findings 6 BENIGN NEOPLASMS A tumor is said to be benign when its microscopic and gross characteristics are considered to be relatively innocent, implying that it will remain localized, it cannot spread to other sites, and is generally amenable to surgical removal. MALIGNANT NEOPLASMS A tumor is said to be malignant when the lesion possesses the ability to invade and destroy adjacent structures, and spread to distant sites (metastasize) and to cause death. BENIGN AND MALIGNANT NEOPLASMS The Good and The Bad? Most people think that benign neoplasms are good, and that malignant neoplasms are bad… …most people are wrong! All tumors are bad, and some are worse than others. 7 MORBIDITY AND MORTALITY OF BENIGN NEOPLASMS Problems associated with benign neoplasms depend upon several important factors: Size of the Tumor Location of the Tumor Secondary Consequences Consider several examples… TUMORS OF THE BRAIN Ependymoma of the brainstem Glioma of the brainstem Meningioma Medulloblastoma of the brainstem HEMANGIOMA OF THE LIVER Hemangiomas represent benign lesions formed by endothelial cells. These can be dangerous due to their size and their tendency to rupture. 8 BENIGN AND MALIGNANT NEOPLASMS Distinguishing Characteristics Cellular Differentiation Anaplasia Rate of Growth Local Invasion Metastasis BENIGN AND MALIGNANT NEOPLASMS Cell Differentiation and Anaplasia Cellular differentiation of tumor cells: • extent to which neoplastic cells resemble their normal counterparts morphologically and functionally Malignant neoplasms that are composed of undifferentiated cells are considered anaplastic. Anaplasia: - to form backward • implies dedifferentiation or loss of the structural and functional differentiation of normal cells BENIGN VERSUS MALIGNANT NEOPLASMS Benign Malignant General Characteristics • Slow growth rate • No metastatic disease • Produces local effects General Characteristics • Rapid growth rate • Metastases are common • Can cause local and/or distant pathophysiological effects 9 CHARACTERISTICS OF MALIGNANT CELLS Differentiation and Anaplasia Examples of cellular pleomorphism in a mammary carcinoma Pleomorphism - variation in size and shape Stromal Response - activity in the supporting connective tissue, “scirrhous” CHARACTERISTICS OF MALIGNANT CELLS Excessive and Abnormal Mitosis BENIGN VERSUS MALIGNANT NEOPLASMS – Each benign or malignant neoplasm will not fit all criteria for each designation – Some neoplasms will have features of the opposite designation 10 TUMOR INVASION AND METASTASIS Cancers grow by progressive infiltration, invasion, destruction, and penetration of the surrounding tissue. Next to the development of distant metastases, local invasiveness is the most reliable feature that distinguishes malignant from benign tumors. TUMOR GROWTH 10 12 •number of •cancer cells diagnostic threshold (1cm) 10 9 time undetectable cancer detectable cancer limit of clinical detection host death 11 THEORETICAL TUMOR KINETICS Tumor kill (%) Surviving cells Viable mass untreated 90 (1-log) 99 (2-log) 99.9 (3-log) 99.99 (4-log) 109 108 107 106 105 Recovery of (doubling time) 1g 100 mg 10 mg 1 mg 100 µg 3.33 days 6.66 days 9.99 days 13.30 days 3 LOG KILL, 1 LOG REGROWTH TUMOR CELL NUMBER Chemotherapy Time PROLIFERATION OF NEOPLASTIC CELLS • Cellular proliferation • Generation Time (T) • Growth Fraction (G) • Proliferation = G x 1/T • Cellular loss • Apoptosis • Necrosis 12 ANALYSIS OF CELL PROLIFERATION M G0 Ki-67 G2 PCNA G1 S increasing Mitotic Index G1 BrdU ANALYSIS OF CELL PROLIFERATION CLINICAL EFFECTS OF NEOPLASMS Tumor Invasion and Metastasis The Major Cause of Cancer Morbidity and Mortality Tumor metastasis can result in major clinical consequences and complications. Loss of Tissue Function Loss of Nervous Control Pain and Discomfort 13 LOSS OF TISSUE FUNCTION Impaired respiration due to lung metastasis Impaired liver function due to liver metastasis Intestinal adenocarcinoma metastatic to lung (left) and liver (right). LOSS OF TISSUE FUNCTION site is very important, maybe more important than size LOSS OF NERVOUS CONTROL Loss of voluntary motor function due to metastasis to major nerves and loss of cognitive function due to metastasis to the brain Histologic appearance of metastatic mammary carcinoma invasive to a peripheral nerve (left) and metastazising to the brain (center, right). 14 PAIN AND DISCOMFORT Related to specific sites of metastasis… Bone Metastasis Systemic Effects of Neoplasms Cancer cachexia CANCER CACHEXIA a wasting syndrome characterized by progressive loss of body fat and lean body mass, accompanied by profound weakness, anorexia, and anemia Tumor size and extent of tumor spread generally correlates with severity of cachexia 15 CANCER CACHEXIA Inadequate caloric intake Changes in energy metabolism Increased catabolism of building blocks (altered sensitivity to insulin) Neoplastic cells may release substances which encourage skeletal muscle breakdown TNF-α ("cachectin") release from macrophages encourages lipolysis (inhibits lipoprotein lipase) Competion of neoplastic cells for nutrients with host cells not a significant contributor to cachexia PARANEOPLASTIC SYNDROMES symptom complexes (other than cachexia), that cannot be readily explained by local or distant spread of the tumor Paraneoplastic syndromes may … ...represent the earliest manifestation of an occult neoplasm ...present significant clinical problems and can be lethal …mimic metastatic disease, confounding treatment ENDOCRINOPATHIES Neoplastic cells produce hormones normal for the cell of origin Pituitary neoplasm - ACTH leading to Cushing’s disease Pancreatic islet neoplasm - insulin production leading to hypoglycemia Sertoli cell tumor in testicle - estrogen production leading to feminization 16 Adrenal-associated Endocrinopathy On the adrenal cortical cells are LH receptors that are activated after neutering by high LH levels Adrenal-associated Endocrinopathy Adrenal-associated Endocrinopathy steroidogenic factor-1 steroidogenic acute regulatory protein 17 Adrenal-associated Endocrinopathy Paraneoplastic Syndromes Paraneoplastic Syndromes 18 Paraneoplastic Syndromes ENDOCRINOPATHIES Neoplastic cells produce peptides which mimic a hormone not produced by the cell of origin “Hypercalcemia of malignancy” Lymphoma and apocrine adenocarcinoma of the anal sac Can lead to osteolysis and soft tissue mineralization 19 Paraneoplastic Syndromes Paraneoplastic Syndromes Miscellaneous: Hypertrophic Osteopathy CANCER STATISTICS How many people alive today have ever had cancer? Since 1990, over 18 million new cases of invasive cancer have been diagnosed. 9.8 million Americans with a history of cancer were alive in January 2001… ...some cancer-free, …others with ongoing disease. 20 COST OF CANCER For 2004, the NIH estimated $189.8 billion… $69.4 billion for direct medical costs $16.9 billion indirect morbidity (lost productivity) $103.5 billion indirect mortality costs (lost productivity) Numbers of Cases/Year COMPARISON OF CANCER RATES IN HUMANS, DOGS AND CATS 4500000 Human Dog Cat 3500000 2500000 1500000 500000 0 All Sites Oral Cavity Mammary Lymphoma WHO GETS CANCER? In general… old people and animals get cancer… 21 AGE-DEPENDENCE OF CANCER 30 25 Cancers at all Sites Incidence Rate 20 15 10 5 0 <20 20-34 35-44 45-54 55-64 65-74 75-84 85+ 1800 1600 1400 1200 1000 800 600 400 200 0 Cases/100,000 Total Cases (%) 35 Cancer is primarily a disease of old age… Are there exceptions to this statement? AGE-DEPENDENCE OF CANCER Potential reasons: Defective DNA repair Decreased immunocompetence Defective regulation of cell proliferation Cumulative exposure to carcinogens HEREDITARY FACTORS The biggest single cause of death was cancer (44%), in particular, osteosarcomas (22%) Boxers have a high incidence of mast cell tumors (among others) The mean survival time is 7 years, the main cause of death is malignant histiocytosis 22 SKIN PIGMENTATION Solar-induced cutaneous neoplasms Melanomas in gray horses GENDER Certain neoplasms are more common in one gender than the other Hormonal dependency Estrogen receptors in mammary neoplasia Androgen-dependency of canine perianal gland neoplasms and prostatic neoplasia/hyperplasia FACTORS CONTRIBUTING TO CANCER INCIDENCE Genetics Gender Race/Ethnicity Habitual Exposures Cultural Practices Occupational Susceptibility to Exposures Cancer Development World Region Environmental Exposures 23 WHAT CAUSES CANCER? Wrath of God? Bad humors? Defective immunity? Viruses? Other infectious agents? WHAT CAUSES CANCER? Circa 1550 BC Ebers Papyrus first major collection of medical observations made on human Neoplasms The Papyrus Ebers: The Greatest Egyptian Medical Document B. Ebbell (1937), Levin and Munksgaard, Copenhagen WHAT CAUSES CANCER? Hippocrates and Galen, 300 BC – 200 AD Both regarded cancer to be a side effect of melancholia (extreme depression characterized by tearful sadness and irrational fears). Cancer results from an imbalance between the black humor (from the spleen) and the other three bodily humors (blood, plegm, and bile). These were the first to attribute cancer to natural causes. 24 WHAT CAUSES CANCER? In the Dark Ages “Cancer families,” “cancer houses,” and “cancer villages” were described. Environmental (infectious) causes for cancer? Genetic predisposition to cancer development? WHAT CAUSES CANCER? Circa 1490-1560 George Agricola Described 1879 as Theophrastus Paracelsus bronchiogenic carcinoma. Described the “Mountain Sickness” or Bergkrankeit (cough, chest pain, shortness of breath), that was frequently suffered by the metal miners of Schneeberg and Joachimsthal (in the Ore Mountains seperating Saxony and Bohemia). WHAT CAUSES CANCER? 1775 Sir Percival Pott Suggested a link between the high incidence of scrotal cancer in chimney sweeps and their exposure to “soot.” P. Pott (1775), Chirurgical Observations Relative to the Cataract, the Polypus of the Nose, the Cancer of the Scrotum, the Different Kinds of Ruptures, and the Mortification of the Toes and Feet. Hawes, Clark, and Collins, London 25 WHAT IS CARCINOGENESIS? The pathogenic process or processes that constitute “carcinogenesis” represent the mechanism or mechanisms of cancer induction. WHAT IS A CARCINOGEN? “Carcinogens” are agents that drive the process of carcinogenesis. …carcinogens cause cancer CARCINOGENESIS MODELS Multistage models of carcinogenesis were proposed to explain the observation that the age-specific incidence curves of many common cancers increase roughly with a power of age. Nordling, C.O. (1953) A new theory of the cancer inducing mechanism. Br. J. Cancer 7:68-72. Armitage, P. and Doll, R. (1954) The age distribution of cancer and multistage theory of carcinogenesis. Br. J. Cancer 8:1-12. 26 CARCINOGENESIS IS A MULTISTAGE PROCESS Chemical Endogenous Radiation Factors Virus Genetic Change Normal Cell Selective Clonal Expansion Initiated Cell Genetic Change Preneoplastic Lesion Malignant Tumor C.C. Harris (1991) Chemical and physical carcinogenesis: Advances and perspectives for the 1990s. Cancer Res. 51:5023s-5044s. CARCINOGENESIS IS A MULTISTAGE PROCESS What are the various stages of carcinogenesis? Initiation Promotion Conversion Progression What is the biological significance of each stage? How were these stages defined? CARCINOGENESIS IS A MULTISTAGE PROCESS Initiation An rapid and typically irreversible process whereby a carcinogen (chemical or other) produces permanent changes in the DNA of target cells (requires cell replication to make it permanent). Initiating agents directly interact with cellular DNA. (Binding of compound to DNA causes mutation) No morphologic hallmarks of initiation 27 CARCINOGENESIS IS A MULTISTAGE PROCESS Promotion The process through which tumor formation is stimulated in tissues that have been exposed to an initiating carcinogen (chemical or other). Promoting agents do not directly interact with cellular DNA. Process occurs over a long period of time (latency period) through a series of usually reversible tissue and cellular changes before the appearance of the first cancer cell. Effects are cumulative (achieve a “threshold” of promotion). CARCINOGENESIS IS A MULTISTAGE PROCESS Progression The process through which the cells of a tumor evolve in a stepwise fashion into more aggressive forms with increasingly malignant behavior. Concept that proliferating cells become progressively more heterogeneous and progressively more “neoplastic”. Carcinogens act as initiators, promoter, or progressors, or any combination of the above. Initiation Initiating Clonal expansion Event 2nd Critical Event Promotion Clonal Expansion nth Critical Event Clonal Expansion Modified from Swenberg et al., 1987. Progression 28 CARCINOGENESIS IS A MULTISTAGE PROCESS Initiation Promotion Conversion Progression Insult Genetic Change Normal Cell Selective Clonal Expansion Genetic Change Genetic Change Initiated Preneoplastic Malignant Cell Lesion Tumor Genetic Change Clinical Cancer Advanced Clinical Cancer INITIATING AND PROMOTING AGENTS IN CHEMICAL CARCINOGENESIS Initiation alone is not sufficient for tumor formation. Initiating agents are not complete carcinogens. Promoting agents can induce tumors from initiated cells, but are not carcinogenic by themselves. Promoting agents are not complete carcinogens. Complete carcinogens act as both initiating agent and promoting agent. METABOLIC ACTIVATION OF CHEMICAL CARCINOGENS Direct-acting carcinogens require no metabolic activation, while indirect-acting carcinogens (or procarcinogens) require metabolic conversion to produce the ultimate carcinogen. Direct-acting and other ultimate carcinogens are highly-reactive electrophiles that can react with nucleophilic (electron-rich) sites in the cell. e.g. benzo[a]pyrene (BP) 29 PROMOTERS OF CHEMICAL CARCINOGENESIS Examples of Promoting Agents Phorbol esters, hormones, phenolic compounds, and drugs Initiated cells respond selectively to promoting agents, while normal cells do not respond. Promoting agents promote cellular proliferation and clonal expansion of initiated cells. CARCINOGENESIS IS A MULTISTAGE PROCESS How were these stages defined? Berenblum, I. and Shubik, P. (1947) A new quantitative approach to the study of the stages of chemical carcinogenesis in the mouse’s skin. Br. J. Cancer 1:383-391. Dimethylbenz[a]anthracene (DMBA) and 12-O-tetra-decanonylphorbol-13-acetate (TPA) DEFINING INITIATION AND PROMOTION Mouse Skin Carcinogenesis Model Initiator = no tumor T T Initiator + Promotor = tumor develops Initiator + Delay + Promotor = tumor develops Promotor = no tumor Promotor + Initiator = no tumor = Initiator = Promotor T= Tumor 30 EXAMPLES OF CHEMICAL CARCINOGENESIS 1930 Specific chemicals (dibenzanthracene) are carcinogens in experimental animals. E.L. Kennaway and I. Heigler (1930) Carcinogenic substances and their fluorescence spectra. Br. Med. J. 1:1044-1046. EXAMPLES OF CHEMICAL CARCINOGENESIS Aflatoxin B1 AFB1 is a fungal toxin that contaminates certain food products such as corn and rice, and acts as a potent hepatocarcinogen EXAMPLES OF PHYSICAL CARCINOGENESIS Ultraviolet Radiation Asbestos UV-light exposure is strongly correlated with development of skin cancer (basal cell and squamous cell carcinoma, melanoma) The characteristic tumor associated with asbestos exposure is malignant mesothelioma of the pleural and peritoneal cavities 31 EXTERNAL FACTORS ASSOCIATED WITH NEOPLASIA Hiroshima, Japan Nagasaki, Japan August 6, 1945 August 9, 1945 1952 Increased incidence of leukemia in Japanese survivors of the atomic bomb blasts. J.H. Folley, W. Borges, and T. Yamawaki (1952), Am. J. Med. 13:311-321. Incidence of leukemia in survivors of the atomic bomb in Hiroshima and Nagasaki, Japan. OCCUPATIONAL EXPOSURE 1974 The first cases of angiosarcoma of the liver in workers exposed to vinyl chloride were described. 1929 Martland and Humphries reported a high incidence of osteogenic sarcomas in watch dial painters chronically exposed to radium. JL Creech, MN Johnson (1974) Angiosarcoma of the liver in manufacture of polyvinyl chloride. J. Occup. Med. 16: 150-151. HS Martland, RE Humphries (1929) Osteogenic sarcoma in dial painters using luminous paint. Arch. Pathol. Lab. Med. 7:406-417. Benzo[a] pyrene Rate Per 100,000 Population HABITUAL EXPOSURE 80 70 60 50 Male Patients Lung Stomach Prostate Colorectum 40 30 20 10 0 1930 1940 1950 1960 1970 1980 1990 Benzo[a]pyrene is found in tobacco smoke and is suspected to be important in the etiology of lung cancer Vincent van Gogh, 1886 E.L. Wynder and E.A. Graham (1950) Tobacco smoking as a possible etiologic factor in bronchiogenic carcinoma. A study of six hundred and eighty-four proved cases. JAMA 143:329-336 32 Increased Numbers of Women Smokers Contributes to the Rise in Lung Cancer Incidence Among Women “…You’ve come a long way, baby!” EXAMPLES OF HORMONAL CARCINOGENESIS Estrogens Androgens Hormone-dependent tumors of the female include mammary carcinoma and adrenal cortical carcinoma (and others) Hormone-dependent tumors of the male include perianal gland tumors and prostatic carcinoma (and others) VIRAL CARCINOGENESIS Peyton Rous produced sarcomas in chickens using a cellfree filtrate prepared from transplantable sarcoma cells. P. Rous (1911) A sarcoma of the fowl transmissible by an agent separable from the tumor cells. J. Exp. Med. 13:397-411. 33 RETROVIRUSES 1 2 • • • 1) 2) 3 3) 4 4) 5 5) 6) include all oncogenic RNA viruses reverse transcriptase (RNA-dependent DNA polymerase) characteristic of group Life cycle Virus attaches to host cell and fuses with membrane Reverse Transcriptase transcribes RNA genome to DNA DNA copy is integrated into host cell’s genome (provirus) Provirus is transcribed into RNA, which is translated into protein Viral proteins assemble into new virion Virion buds from infected cell 6 CONTINUUM OF RETROVIRUS-MEDIATED EVENTS TO TRANSFORMATION OF DISEASE Activation Immortalization Early Viral Effects Polyclonal Proliferation Transformation Clinical Genetic Complications Mutations NOD/SCID of ATL Late ViralModel Effects Monoclonal Proliferation Paraneoplastic Events e.g. bone lysis, hypercalcemia Retrovirus Infection and Replication Dysregulation of Cell Growth Control and Altered Cell Interactions Retrovirus Alteration of Cell Regulation and Signaling Tumor viruses transform infected cells • • • • • • Rous sarcoma virus carries src gene that causes cell transformation c-src plays a role in cellular processes in normal cells, but v-src is able to transform normal cells to tumors = oncogene ALV can replicate without src gene proviral DNA becomes integrated into host DNA next to a c-src gene provirus and c-src are co-transcribed into hybrid RNA after splicing out c-src introns, the hybrid is packaged into a virus particle that may be ancestor to RSV How do viruses without oncogene transform cells? 34 How do viruses without oncogene induce tumors? • • • • • • slowly tranforming retroviruses activate proto-oncogene by inserting their genomes adjacent to cellular genes numerous B-cell lymphomas are produced by ALV that have their provirus integrated into chromosomal DNA segment that carries c-myc proto-oncogene integrated between first noncoding exon of c-myc gene and the second exon where the reading frame of c-myc begins insertions occur randomly transcription of c-myc through the strong constitutively acting ALV promoter Myc drives cell proliferation and activated c-myc gene will multiply uncontrollably EXAMPLES OF RETROVIRAL NEOPLASMS Avian Leukosis-sarcoma virus Feline Leukemia-sarcoma virus DNA viruses - some contain viral oncogenes - others alter host proto-oncogenes - Herpesviruses - Papillomaviruses - Poxviruses SV40 Shope 35 Tumor viruses induced change in cell phenotype • • • • • • • • altered morphology loss of contact inhibition anchorage independence immortalization require fewer mitogenic factors high saturation density unresponse to anti-growth signals increased transport of glucose • viral genome becomes integrated in host cell DNA • sucrose gradient sedimentation: • blue = cellular DNA, green = viral DNA, red = SV40 in cellular DNA EXAMPLES OF DNA VIRUS NEOPLASMS • Herpesvirus – Marek’s disease induced lymphosarcoma in chickens – Lymphosarcoma in rabbits, guinea pigs, and non-human primates – Lucke’s virus - renal carcinoma in frogs – Epstein-Barr virus - Burkitt’s lymphoma and nasopharyngeal adenocarcinoma in humans EXAMPLES OF DNA VIRUS NEOPLASMS • Poxviruses • Shope fibroma virus - fibromas in rabbits • Yaba pox - histiocytoma in rhesus monkeys R.E. Shope (1933) Infectious papillomatosis of rabbits. J. Exp. Med. 58:607-624. 36 EXAMPLES OF DNA VIRUS NEOPLASMS • Papilloma viruses • Associated with hyperplastic and neoplastic growths • Found in a variety of species • Equine sarcoid Papillomaviruses • L1 and L2 capsid proteins – late stage structural proteins – highly conserved • DNA – one strand encodes open reading frames – consists of 7 genes (E1-E7) – deletion of E2 leads to derepression of main promoter – E6 and E7: main transforming proteins • repression leads to loss of oncogenic potential • immortalize keratinocytes Papillomaviruses • Viral capsid genome encoded late and only in terminally differentiated epithelial cells • Capsid viral antigen and virions only detected in keratinized cells • replicates in the host nucleus - remains episomal (replicates intranuclear, but extrachromosomally) 37 Bovine Papillomaviruses Bovine Papillomaviruses and Bracken-fern Feline Papillomaviruses 38 Feline Papillomaviruses Feline Papillomaviruses GENETICS AND CANCER Theodor Boveri “…a particular, incorrect chromosome combination which is the cause of abnormal growth characteristics passed on to daughter cells…” T. Boveri (1914) Zur Frage der Entstehung Maligner Tumoren (“The Origin of Malignant Tumours”) Gustav Fischer Verlag, Jena 39 CANCER IS A GENETIC DISEASE DNA is the Biological Target of Carcinogenesis Are all carcinogens mutagens? Are all mutagens carcinogens? DNA and bound carcinogen (left) and chemical adduct (right). CANCER IS A GENETIC DISEASE Mutagens cause changes in the base composition of the DNA Carcinogens cause cancer Is mutation necessary for cancer induction? GENETIC BASIS OF NEOPLASIA Epigenetic Mechanisms: Alteration in gene expression without altered DNA Affect transcription or translation Genetic Mechanisms: Direct interaction with DNA Evidence for somatic mutation Most carcinogens are mutagens Chromosomal abnormalities common in neoplasms RESULT Abnormal gene products Abnormal quantities of normal gene products “De-repression” (reactivation) of embryonic genes 40 SIMPLE GENETIC DISEASE Simple genetic diseases are caused by inherited mutations in a single gene that are necessary and sufficient to determine the phenotype. For example: Duchenne Muscular Dystrophy Dystrophin Mutation Muscular Dystrophy COMPLEX GENETIC DISEASE Complex genetic diseases involve single gene mutations that can predispose patients to pathological conditions, but the defective gene itself is not sufficient to guarantee development of disease. For example: Atherosclerosis LDL Receptor Mutation Lipid Accumulation Atherosclerosis CANCER AS A DISEASE STATE “Cancer is, in essence, a genetic disease.” Cancers become manifest following the accumulation of a critical number of genetic (and epigenetic) alterations, in response to imperfections in the DNA replication machinery or through DNA damage caused by environmental mutagens. Ultimately…. CANCER IS A DISEASE OF ABNORMAL GENE EXPRESSION. From: B. Vogelstein and K.W. Kinzler (2002) Introduction. In: The Genetic Basis of Human Cancer, Second Edition (B. Vogelstein and K.W. Kinzler, Eds), New York, McGraw-Hill, pp. 3-6. 41 CANCER A Disease of Abnormal Gene Expression CANCER PATHOGENESIS “The development of cancer in humans involves a complex succession of events that usually occur over many decades. During this multistep process, the genomes of incipient cancer cells acquire mutant alleles of proto-oncogenes, tumor suppressor genes, and other genes that control, directly or indirectly, cell proliferation.” W.C. Hahn and R.A. Weinberg (2002) Rules for making human tumor cells. New England Journal of Medicine 347:1593-1603. WHAT ARE CANCER GENES? “…proto-oncogenes, tumor suppressor genes, and other genes that control, directly or indirectly, cell proliferation.” W.C. Hahn and R.A. Weinberg (2002) Rules for making human tumor cells. New England Journal of Medicine 347:1593-1603. 42 WHAT ARE CANCER GENES? …Genes that cause cancer… This would include classically defined… Viral Oncogenes Cellular Proto-oncogenes Tumor Suppressor Genes WHAT ARE CANCER GENES? …Genes that contribute to cancer… There are a number of genes and classes of genes that contribute to neoplastic transformation that do not conform to the definition of classic oncogenes or classic tumor suppressor genes. GATEKEEPERS AND CARETAKERS GATEKEEPERS …Genes that cause cancer… genes that directly regulate the growth of tumors by inhibiting their growth or by promoting their death CARETAKERS …Genes that contribute to cancer… genes that encode proteins that do not directly regulate tumor growth, products of caretaker genes promote genetic stability K.W. Kinzler and B. Vogelstein (2002) Familial cancer syndromes: The role of caretakers and gatekeepers. In: The Genetic Basis of Human Cancer, Second Edition (B. Vogelstein and K.W. Kinzler, Eds), New York, McGraw-Hill, pp. 209-210. 43 GATEKEEPERS Tumor Suppressor Genes Cancers Associated with Defects in Tumor Suppressor Genes Retinoblastoma (Rb1) Li-Fraumeni Syndrome (p53) Wilms’ Tumor (WT1) Hereditary Breast Cancer (BRCA1 and 2) And others…. CARETAKERS DNA Repair Genes Hereditary Cancer Syndromes Related to Defective DNA Repair Xeroderma pigmentosum Ataxia-telangiectasia Bloom Syndrome Hereditary Nonpolyposis Colorectal Cancer (hMSH2, hMLH1, hPMS2, hPMS1) GATEKEEPERS AND CARETAKERS Normal Gatekeeper Pathway Caretaker Pathway Neoplasia How do Caretakers and Gatekeepers contribute to carcinogenesis, individually, or in combination? Mutation of a Mutation of 2nd Mutation of a Mutation of 2nd caretaker gatekeeper caretaker gatekeeper gene allele gene allele gene allele gene allele leads to leads to genetic instability tumor initiation 44 What is the contemporary definition of cancer gene? Cancer genes encode for positive mediators and negative mediators of neoplastic development. CANCER GENE CLASSIFICATION Proto-oncogenes Tumor Suppressor Genes Caretakers Gatekeepers Positive Mediators Negative Mediators Cancer genes encode for positive mediators and negative mediators of neoplastic development. COMMON MUTATIONAL MECHANISMS IN CANCER GENES Chromosomal Abnormalities Large-Scale Deletions Amplifications Numerical Abnormalities Rearrangements Sequence Alterations Point Mutations Insertions Deletions 45 CHROMOSOMAL ALTERATIONS Chromosomal alterations that represent nonlethal genetic damage can lead to carcinogenesis. Most chromosomal alterations represent structural aberrations that produce various functional consequences. Some chromosomal alterations are without biological consequence. BIOLOGICAL CONSEQUENCES GENE/PROTEIN ACTIVATION Gain of gene/protein function Loss of gene/protein regulation GENE INACTIVATION/LOSS OF PROTEIN FUNCTION Loss of gene expression Loss of normal protein function EXAMPLES c-Ras Proto-oncogene p53 Tumor Suppressor Gene Functional Capabilities of Cancer Cells Hanahan and Weinberg (2000). Cell 100: 57-70. 46 SELF-SUFFICIENCY IN GROWTH SIGNALS PROTO-ONCOGENES CARETAKERS POSITIVE MEDIATORS SELF-SUFFICIENCY IN GROWTH SIGNALS Oncogene: a cancer-causing gene of viral origin Proto-oncogene: cellular homologue of a viral oncogene Oncogene: any cancer-causing gene Proto-oncogene: normal counterpart of an activated oncogene Positive mediators of neoplastic development include oncogenes, as well as other cancer-promoting genes ONCOGENES More than 20 viral oncogenes have been identified, each of which has a corresponding cellular proto-oncogene v-sis, v-raf, v-rel, v-ski, v-erbA, and others Cellular Proto-oncogenes Additional cellular proto-oncogenes have been identified (without a prior viral counterpart) More than 100 cellular proto-oncogenes have been identified…. 47 Can cancers be triggered by the activation of endogenous retrovirus ? • New model in the mid 1970s explained how tumor viruses could participate in formation of cancer: Based on retrovirus biology • Endogenous retroviruses can explain tumor development in the absence of infectious retroviral spread • Infection of gonads can result in integration of retroviral provirus into cell chromosomes • Provirus becomes ensconced in genome of descendants: endogenous retrovirus • Activation of latent viruses can lead to malignancy (leukemia, AKR mouse) Human ERV • Probing genomic DNA with DNA of infectious retrovirus to detect ERV • Each band in a gel channel represents a restriction fragment in a cell genome that carries part or all of an ERV genome • The variability of ERV integration sites between lab strains indicates that numerous ERV’s have been integrated into the germ line since the seperation from Mus musculus • In contrast to mouse ERV genomes, human ERV genomes detected in human DNAs show rather similar integration sites across the species: germ-line integration before emergence of human species • Polymorphic differences are largely the results of recombination between terminal LTR sequences at the end of individual ERV proviruses xenotropic murine retroviruses Origin of endogenous retroviruses Does this model apply to humans ? • No tumor viruses in the majority of human cancer • Disease-inducing retroviruses are disadvantageous and usually eliminated, most ERV are transcriptionally silent • No reports of infectious retroviral particles in human tumors have been verified • Most human ERV are relics of ancient germ-line infections • HERV-K entered human germ line relatively recently, its proviruses are intact, but not associated with cancer • Thus, suspected that most of human cancer could be induced by mutagens which mutate normal growthcontrolling genes such as cellular oncogenes 48 DNA transfection assay is a new strategy for detecting non-viral oncogens • • • • Do transformed cells carry mutated genes that function as oncogenes? Introduction of DNA from chemically transformed cells into normal cells by transfection and determination whether these recipient cells become transformed NIH3T3 cells (mouse fibroblasts) contact-inhibited, non-tumorigenic Changes in cell morphology, loss of contact inhibition Oncogenes discovered in human cell lines are related to those carried by transforming retroviruses Homology between transfected oncogenes and retroviral oncogenes • DNA probe derived from H-ras oncogene present in Harvey rat sarcoma virus formed hybrids with oncogene detected in NIH 3T3 cells transformed by transfection with DNA from bladder carcinoma cells 49 DETECTION OF FIRST HUMAN ONCOGENE (EJ-RAS) Tumorigenicity Assay High Molecular Weight DNA From Tumor Cells Human Bladder Carcinoma Cells Transfect 2-3 Weeks Mouse 3T3 Fibroblasts Focus of Transformed Cells (Neoplastic?) How are proto-oncogenes activated? • • • • Structural alteration or mutation Overexpression Deregulated expression All or some of the above bcr-abl Translocations producing a fusion protein • abl gene (Abelson murine leukemia virus), rapidly tumorigenic retrovirus, maps to chromosome 9q34 • Fused with sequences clustered at 22q11, called breakpoint cluster region = bcr (both are multidomain, multifunctional proteins) • Identified in 95% of Chronic myelogenous leukemia (CML) • Different breakpoints in different leukemias 50 Consequences of chromosome rearrangement The c-abl proto-oncogene encodes a tyrosine kinase The Philadelphia chromosome in CML t(9,22)(q34;q11.1) • The Philadelphia chromosome translocation results in activation of the c-abl proto-oncogene through production of the bcr/abl fusion gene • Expression of the bcr/abl gene is driven by the bcr gene promoter, and the transcript produced represents a chimera consisting of the 5’ portion of the bcr gene, and the 3’ portion of c-abl • The bcr/abl protein expresses enhanced tyrosine kinase signaling activity compared to the normal ABL protein (gain of function) • Oncogenic activity results from inappropriate expression or overexpression of the proto-oncogene product H-ras point mutations • • • Point mutation by chemical carcinogenes Genes activated in chemically induced tumor were identified by DNA transfection assay In human tumors, the activated ras genes by single point mutations show a mutation in codon 12, 13, 59, and 61 A list of point mutated ras oncogenes carried by a variety of tumor cells H-ras oncogene activation Identification of the transforming region of the EJ-ras gene Normal Cells Cancer Cells Transfection 2-3 Weeks 2-3 Weeks 51 Identification of the transforming region of the EJ-ras gene Normal EJ-ras Recombinants DNA region responsible for transforming activity Consequences of point mutations Protein function of the c-H-ras proto-oncogene product requires interaction with GTP • Point mutational alteration of a proto-oncogene sequence results in a change of amino acid in the protein structure, typically reflecting a nonconservative change. • These amino acid changes can alter protein function. • Oncogenic activity results from gain of function. • In the case of c-ras, point mutation results in loss of GTPase activity, resulting in constitutive activation of the signaling function of RAS. Detection of proto-oncogenes in increased copy numbers in human tumor cell genomes • Proto-oncogene amplification • Detection of cellular protooncogenes in multiple copies in various tumors and transformed cell lines • Increased expression of amplified proto-oncogenes : • a role in the development and progression of these tumors • Correlated with decreased survival in breast cancer Amplification of erbB2/neu in breast cancers 52 Elevation of expression of 17q genes together with overexpression of HER2/Neu/erbB2 • Gene amplification can be difficult to interpret • HER2 protects cells from apoptosis and induces growth and division • Amplification of HER2 occurs as consequence of the amplication of an entire chromosomal segment – an amplicon (encompasses additional genes) • Amplification of other genes influences tumor phenotype Variation on the theme: the myc oncogene • the myc oncogene can arise by multiple distinct mechanisms • myc amplification in childhood neuroblastomas by increasing copy number • Detection of multiple copies through ISH • Homogenous staining regions (HSRs) contain multiple copies of N-myc encompassing gene • broken away from normal site and are associated with different chromosomal regions • High copy numbers are associated with poor prognosis Variation on the theme: the myc oncogene - Chromosomal translocation of c-myc in Burkitt’s lymphoma, placed under control of a transcription-controlling sequence of immunglobulin gene - Three additional chromosomal translocations involving chromosome 8 - Malarial parasites and EBV virus are an etiological factor ? - Immunosuppression predisposes to EBV? B lymphocytes are immortalized by EBV and proliferate continously Enzymatic machinery dedicated to organizing normal Ig gene arrangement misfires Inappropriate juxtaposition of Ig gene and c-myc oncogene 53 Oncogenic activity of Myc overexpression • Myc belongs to family of basic helix-loophelix (bHLH) transcription factors • act as heterodimers: Myc-Max × transcription • Myc replaced by Mad during differentiation • Fusion of Myc to ER > binding of ligand (estrogen) > Myc-ER migrates into nucleus > associate with Max and advance cell into G1/S Cooperation Between Oncogenes c-myc Primary Fibroblasts c-ras NIH 3T3 Primary Fibroblasts NIH 3T3 c-myc + c-ras Primary Fibroblasts NIH 3T3 Cooperation Between Oncogenes Mutant Ras can generate tumors in transgenic mice % Tumor Free Mice MMTV-Ras DNA Myc Ras Myc + Ras 0 100 Age (days) 200 54 ONCOGENES • • • • • Normal proto-oncogene precursor is associated with cell growth or cell death signaling Mutated or overexpressed consistently in certain human or animal tumor types Mutated or overexpressed oncogene form can be shown to augment growth signaling or cell cycle progression when introduced into normal cells in culture The mutated or overexpressed oncogene can transform normal rodent cells into cancerous cells by itself or in conjunction with another oncogene The oncogene can cause tumors in mice when overexpressed as a transgene POSITIVE MEDIATORS OF NEOPLASIA Neoplastic disease is characterized by uncontrolled cell proliferation Positive mediators of neoplastic development function to promote cell cycle progression and cell proliferation POSITIVE MEDIATORS OF NEOPLASIA Positive Growth Factors EGF, TGF-α, PDGF-β, and others Growth Factor Receptors EGFR, PDGFR, C-KIT and others Signal Transduction K-RAS, BRAF, β-catenine Nuclear and Cell Cycle Regulation C-MYC, CYCLIN D and E, CDK4 55 FUNCTIONAL SUBCELLULAR LOCALIZATION OF PROTO-ONCOGENE PRODUCTS Growth • Highly conserved in evolution Growth Factors Factors Receptors • Important regulators of normal cell growth and differentiation GTPase • Maintain the ordered Proteins progression of the cell Cytoplasmic Serine-Threonine cycle, cell division, and Guanine Kinases Nucleotide differentiation (lost in cancer) Exchange • Mutation that alter the Proteins structure, levels, or sites of Nuclear expression of the gene Transcription Factors products > activation of Cytoplasmic Membrane-associated oncogenic potential Tyrosine Kinases Growth signal transduction • Most proto-oncogene products are components of growth signal transduction pathways. • The signal often begins at the cell surface, ends in the nucleus, and results in cell cycle entry and progression. • The presence of shaded components in the pathway represent proteins that can be mutated to become oncogenic. • Once oncogenic, such proteins can promote constitutive signaling to downstream components independent of upstream stimuli. Oncogene activated deregulated signaling Cell surface Growth Signal + Upstream Effector + ProtoOncogene Protein + Downstream Effector Regulated Cell Division Normal Regulated Growth Signaling No Cell Surface Growth Signal Oncogene Activated Deregulated Signaling Oncogene Protein + Downstream Effector Continuous Cell Division 56 Growth factor receptors There are a large number of growth factor receptors on the surface of the cell that mediate the initial phase of growth signal transduction, usually through the binding of a growth factor ligand. These receptors can be grouped into two major categories: those with intrinsic tyrosine kinase activity and those without. Many of the receptor tyrosine kinases (RTKs) can be oncogenes. RTKs implicated in cancers are indicated in bold and italics. Ligand binding membrane Tyrosine kinase Receptor tyrosine kinases The key structural domains of receptor tyrosine kinases are: 1. Extracellular ligand binding domain 2. Transmembrane domain 3. Cytoplasmic tyrosine kinase domain - highly conserved 4. Cytoplasmic kinase regulatory domains - usually C terminal Structure of EGFR receptor Receptor tyrosine kinases Y Serine Threonine Kinase P T P S The conservation of the tyrosine kinase domain and its presence in so many of the oncogenic receptors indicate that it is a critical component of the growth signaling response. Kinase Target Protein Y P Y S T P P P Dual Specificity Kinase Phosphorylation of proteins on either tyrosine, serine, or threonine is very common. T S Tyrosine Kinase The Human Genome Project identified about 520 protein kinases. About 90 are tyrosine kinases. Yet tyrosine kinases are disproportionately represented among the oncogenes. Phosphorylation by a kinase can induce: (a) (b) (c) (d) (e) (f) (g) (h) Conformational changes Dissociation of other interacting proteins Reassociation of other interacting proteins Reassociation or dissociation with RNA or DNA Induction of biochemical or enzymatic activity Reduction of biochemical or enzymatic activity Change in cellular localization Initiation or termination of biological processes (e.g. apoptosis, cell cycle progression, etc.) 57 Receptor monomers and dimers Inactive receptor monomers are in equilibrium with inactive and active receptor dimers Binding of growth factor ligand may induce a dimerization of the bound receptors, resulting in stabilization of active dimer formation and an activation of the cytoplasmic tyrosine kinase • • Inactive disulfide bridged insulin-receptor dimers are in equilibrium with active dimers. Insulin binding stabilizes the active dimeric state leading to PTK activation. Variations in receptor dimerization A. B. C. VEGF binds as a dimer to two monomers of its receptor Flt-1 thereby bringing them together each of the FGF-2 ligands binds to the ligand binding domain of a receptor monomer FGF-R1; each ligand-binding domain of the receptor subunits is composed of 2 subdomains; receptor dimerization occurs only if the 2 FGF ligand molecules are bound to a heparin molecule (a glycosaminoglycan component of the extracellular matrix) molecules of TGF-α bind individually to the 2 subunits of EGF-R Growth factor receptor homo- and heterodimerization While the EGF receptor can homodimerize after binding to EGF ligand, it can also form heterodimers with three other members of a family of EGF-like receptors (EGFR/HER/erbB). The ligand specificity (where known) is indicated for each family member. Interestingly, HER3 does not have an active tyrosine kinase domain and must heterodimerize ErbB2 ErbB3 ErbB4 to form an active signaling complex. the erbB receptor consists of three domains: a ligand-binding extracellular domain containing two cysteine-rich regions (CR1 and CR2), a transmembrane domain, and an intracellular domain containing a tyrosine kinase region. Note at left that homodimers of the ErbB/HER family exhibit weaker signaling than do heterodimers, particularly heterodimers Inactive with ErbB2. This has major significance for human breast cancers kinase where ErbB2 is often amplified and overexpressed. Note also the many biological effects that result from heterodimer signaling. 58 Growth factor receptor homo- and heterodimerization Homodimers of the ErbB/HER family exhibit weaker signaling than do heterodimers, particularly heterodimers with ErbB2. This has major significance for human breast cancers where ErbB2 is often amplified and overexpressed. Interactions of ErbB receptors The monomeric structure of ErbB2 (with no ligand) resembles the activated dimer state (with ligand) of the EGF receptor (ErbB1). Thus, ErbB2 is always in a ready state to form dimers without the usual ligand-dependent conformational shifts necessary for the ligand binding family members. Some ErbB family members, such as ErbB2, inefficiently form homodimers and may be dependent on heterodimerization with ErbB3. Once the ErbB2/ ErbB3 heterodimer is formed, signaling is much more efficiently initiated Dimerization of receptors induces tyrosine autophosphorylation Tyrosine autophosphorylation following ligand binding and dimerization - the conformational changes and dimerization of the ligand bound receptor juxtapose the cytoplasmic tyrosine kinase domains and result in trans-phosphorylation of the two monomer cytoplasmic domains. - these phosphorylated tyrosines provide docking sites for downstream signaling targets containing PTB or SH2 domains. 59 Deregulated firing of growth factor receptors - deletion of extracellular domain results in truncated receptor - can be the result of mutation in receptor-encoding gene or alternative splicing of receptor pre-mRNA - as a result receptor emits signal into cell without ligand binding - EGF receptor is capitated in one third of glioblastomas Gene fusion causes constitutively dimerized receptors • • • receptor dimerization occurs in a number of malignant tumors when the genes encoding growth factors become fused to unrelated genes that happen to specify proteins that normally dimerize or form higher order oligomers as a consequence the receptor portion of these hybrid fusion proteins are dragged together by the oligomerizing proteins to which they have been joined the end result is a constitutive ligand-independent dimerization of the affected receptors c-kit proto-oncogene L M N NC 60 Kit receptor activation • juxtamembrane domain (JM) sits above the N-terminal lobe of the two-lobed Kit tyrosine kinase • ligand binding (SCF) to receptor results in dimerization and transphosphorylation of tyrosine residues in JM domain, thereby dissociating it from N-terminal lobe (“moves out of the way”) • results in transphosphorylation of normally obstructing tyrosine residue in catalytic cleft, contributes to kinase activation TGF-β receptor • the TGF-β has a structure that is similar to receptor tyrosine kinases • both signal through cytoplasmic kinase domains • kinase domains of TGF-β receptors phosphorylate serine and threonine, rather than tyrosine residues • type II receptor is brought in contact with type I receptor through TGF-β • phosphorylates and activates kinase carried by type 1 receptor Cytokine receptor • the interferon receptor (IFN-R) carries noncovalently attached tyrosine kinases of the Jak family (Tyk2, Jak1) • upon binding of α-interferon the 2 tyrosine kinases transphosphorylate and activate each other • subsequently they phosphorylate the C-terminal tails of the receptor subunits, thereby inducing the signaling 61 Notch receptor • embodies very primitive type of signaling • after binding ligand, delta, Notch undergoes two successive proteolytic cleavage events • resulting C-terminal fragment is freed and migrates to the nucleus • Notch pathway contributes to Ras-mediated cell transformation • constitutively active mutant forms of Notch fire in ligandindependent fashion and have been found in 50% of adult T-cell leukemia Patch-Smoothened signaling system • hedgehog pathway utilizes its own unique signaling system • Smoothened, a seven-membranespanning surface receptor is normally inhibited by Patched, which contains a 12 membranespanning domains • Gli is cleaved into a protein that moves into nucleus functioning as a repressor of transcription • when the hedgehog ligand binds to Patched, it releases Smoothened, which prevents cleavage of Gli so it can act as an inducer of transcription Mutant forms of Ptc and Smo in basal cell carcinomas Frizzled receptors signaling • receptors of the Wnt proteins are all members of the Frizzled family of transmembrane proteins • with no ligand, Axin and Apc allow glycogen synthase kinase-3β (GSK-3β) to phosphorylate β-catenin • this marks β-catenin for rapid destruction • when wnt binds to Frizzled, the activated receptor via the Dishevelled protein causes inhibition of GSK-3β Tethered to ECM 62 Serpentine receptors • seven-membrane-spanning receptors, serpentine, are associated with heterotrimeric G proteins (α, β, γ subunit) • binding of a ligand, stimulates α subunit to release GDP and bind GTP instead • the α subunit dissociates from the βand γ-subunits • independently, the α as well as the β + γ complex can regulate enzymes that evoke a variety of downstream signals • the signal is halted after the α-subunit hydrolyzes its bound GTP and re-associates with other units • very common receptor that rarely contributes to cancer Integrine receptors • important for growing anchorage dependent • heterodimeric, with α- and β-subunit • associated with ECM through ectodomains and cytoplasm through cytoplasmic domains • β-subunit links to cytoskeleton (actin, talin, vinculin), send signals “inside-out” Integrine receptors • inactivation of β-integrin in mouse mammary tissues • minimal effect on mammary development in controls (A) and knock-outs (B) [β-integrin = red] • transgenic mice (expressing polyomavirus oncogene) prone to mammary tumorigenesis • abundance of pre-malignant hyperplastic nodules (black) versus normal epithelium (red) in β-integrin positive mice • reduction of premalignant foci in knock-out mice (survive, but can’t proliferate) 63 Alternative mechanisms of transformation by Ras • • • • • • ras-transformed cells release growth factors (e.g. TGF-α) act in autocrine fashion (activate EGFR) promoting proliferation co-expression of TGF- α and EGFR in a breast carcinoma “normally” Ras acts “upstream” of the EGF receptor alternatively, Ras can be in the middle of signaling cascade, operating “downstream” of EGFR How is Ras activated “downstream”? Regulation of RAS signaling GTP Operate as binary switches similar to heterodimeric G proteins GTP Active Ras Downstream Signals (Raf1, MAPK, MAPKK, RSK, c-jun) GTP Upstream Signals GDP Guanine nucleotide exchange factors (GEF) Inactive Ras GDP GTPase-activating Proteins (GAP) Functional Consequences of c-H-ras Proto-oncogene Activation GTP GTP Active Ras Downstream Signals (Raf1, MAPK, MAPKK, RSK, c-jun) GTP Upstream Signals GDP Guanine nucleotide exchange factor (GEF) Inactive Ras GDP oncogenic mutation GTPase-activating Proteins (GAP) 64 Cytoplasmic Signaling • • responding to mitogenic signals, growth factors can release a diverse array of biochemical signals transcription of some genes is observed within minutes (immediate early genes) while transcription of others (delayed early genes) occurs with a lag since they require synthesis (can be blocked by cycloheximidine) Tyrosine phosphorylation and SH2 domains • • • • • • Src has 3 homology domains: SH1 harbors the catalytic function SH2 acts as an intracellular receptor for specific phosphotyrosines whose unique identities are determined by the oligonucleotide sequence on their C’ side SH3 recognizes and binds certain proline-rich domains of substrate as a consequence of ligandinduced transphosphorylation, a RTK displays on its cytoplasmic tail an array of phosphotyrosines phosphotyrosines will attract and bind to specifc SH2 domain resulting in downstream signaling non-receptor tyrosine kinase Src • • • • the SH2 group of Src normally binds intramolecular to phosphotyrosine residue at position 527 of the C-terminus this obstructs catalytic cleft (between N- and C-lobe), N-lobe bound to SH3 when activator (PDGF-R) becomes phosphorylated, there is a switch from intramolecular to intermolecular binding (SH2 binds to PDGF-R) SH3 detaches and catalytic cleft is opened for secondary phosphorylation SH3 SH2 SH3 SH2 65 Activation of Ras through SH2 groups • • • • an association of Sos, the Ras guanine nucleotide exchange factor (GEF) with ligandactivated growth factors is achieved by bridging proteins 2 SH3 domains of Grb2 bind to prolin-rich domain of Sos while its SH2 domain binds to phosphotyrosine located on: - C-terminal tail of receptor - Shc, which binds to receptor anchoring Sos to receptor will induce release of GDP from Ras thereby activating it this mechanism explains how growth factors acquire signaling specificity Bridging proteins Grb2 and Shc Jak-STAT pathway • • • • pathway depends on the actions of Jak tyrosine kinases Tyk2 and Jak1, which are attached noncovalently to a number of cytokine receptors (interferon, TPO, EPO) following binding of ligand the 2 tyrosine kinases transphosphorylate and activate each other and subsequently they phosphorylate the C-terminal tails of the receptors, which than attract STAT proteins that bind through their SH2 domain and become phosphorylated STATs dimerize and translocate to nucleus constitutively activated in many cancers (eg melanomas) The Ras-Raf-MAP kinase pathway • • • signaling cascade of overall plan MAPKKK > MAPKK > MAPK most important pathway for transforming powers of Ras oncogene stimulates expression of important growth-regulating genes mutated in 60-70% of melanomas MAPK = mitogen activated kinase activation of promoters of the two immediate early genes encoding the Fos and Jun transciption factors 66 The Ras-PI3 kinase pathway • • • Ras can associate with and activate phosphatidylinositol 3-kinase (PI3K) results in formation of phosphatidylinositol (3,4,5)-triphosphate (PIP3) and thereby activation of Akt/PKB and Rho-GEFs Akt/PKB can suppress apoptosis by blocking Bad and stimulate cell proliferation by blocking GSK-3β (antagonist to cyclin D1 and Myc) The Ras-Ral pathway • • • interactions of ras with Ral-GEF activate the Ral proteins Ral-A and Ral-B GTP-bound ral proteins proceed to activate numerous downstream pathways Cdc42 and Rac enable motility through their effects on cytoskeleton Complexity of the Ras effector pathways 67 INSENSITIVITY TO ANTI-GROWTH SIGNALS TUMOR SUPPRESSOR GENES GATEKEEPERS NEGATIVE MEDIATORS INSENSITIVITY TO ANTI-GROWTH SIGNALS About 20 tumor suppressor genes have been identified… Genetic Linkage Analysis Loss of Heterozygosity (Allelic Deletion) Mutation Analysis Candidate Genes Rb1, WT1, p53, APC, NF-1, NF-2, BRCA1, BRCA2, VHL, p16, DCC, and others TUMOR SUPPRESSOR GENES How did we identify oncogenes? – Identification of retroviral oncogenes – Molecular cloning of oncogenic DNA sequences at chromosomal breakpoints – DNA tranfection assay with DNA derived from cancers • Identification of the TSG is far more difficult. • Why ? 68 EVIDENCE OF TUMOR SUPPRESSOR GENES Somatic cell hybrids generated from tumorigenic and nontumorigenic cell lines (fusion with inactivated Normal Cell Sendai virus) are (Fibroblast) typically not tumorigenic, suggesting that specific chromosomes confer tumor suppression Cell Fusion Tumor Cell Nontumorigenic Cell EVIDENCE OF TUMOR SUPPRESSOR GENES • Wildtype genes antagonize cancer phenotypes > cancer phenotype is “recessive”. • Recessive means loss of both alleles of TSG • Loss of tumor suppressor Virus induced protein function by mutation is lot more easier than hyperactive mutation of oncogenes by mutation • Case against TSG: probability to inactivate a single gene is 10-6 per cell generation, so 10-12 per cell generation for silencing both copies Non virus induced PEDIATRIC RETINOBLASTOMA • Retinoblastoma is the most common intraocular malignancy in children (worldwide incidence between 1 in 13,500 and 1 in 25,000 live births) • Tumor arises from an oligopotential stem cell precursor of multiple retinal cell types • Differences between unilateral versus bilateral retinoblastoma early versus late onset • Why? 69 Unilateral versus bilateral retinoblastoma • • • • Bilateral Retinblastoma: – other types of cancer (osteosarcoma, Ewing sarcoma, leukemia, lymphoma) Unilateral Retinoblastoma: no other tumors Sporadic and hereditary tumors - why? Pedigree shows multiple generations of a kindred afflicted with familial retinoblastomas – confirmation of involvement of genetic alteration by cytogenetic analysis – microscopically visible deletion of one chromosome 13, band q14 – autosomal dominant hereditary form of retinoblastoma : large deletions of chromosome 13 – germ-line mutation in hereditary retinoblastoma – usually of parental lineage (more cell divisions in sperm) Unilateral versus bilateral retinoblastoma • The rate of familiar tumors was consistent with a single random event, while the sporadic tumors behaved as if two random events were required for their formation • Predicted that one of the alleles of the Rb genes in familial Rb carries mutant form 10-6 /cell division 10-12 /cell division • Inherited and somatic changes may collaborate in tumor formation • Linked the notion of recessive genetic determinants for human cancer to the somatic cell genetic studies TWO-HIT HYPOTHESIS FOR RETINOBLASTOMA Female The two-mutation model accounts for the dominant inheritance of susceptibility to retinoblastoma However, it was recognized that the susceptibility gene did not function as a single dominant determinant of neoplastic transformation at the cellular level Normal Male Affected A.G. Knudson, Jr. (1971) Mutation and cancer: Statistical study of retinoblastoma. Proc. Natl. Acad. Sci. U.S.A. 68:820-823 70 HEREDITARY RETINOBLASTOMA Rbmut Sperm Rb Rbmut Rbmut Egg Homozygous Mutant Tissue Rbmut Rb Heterozygous Individual Somatic Mutation Development of Retinoblastoma (usually with multiple tumors) SPONTANEOUS RETINOBLASTOMA Rb Rb Rbmut Rbmut Homozygous Normal Individual Homozygous Mutant Tissue First Somatic Mutation Second Somatic Mutation Rbmut Rb Heterozygous Tissue Development of Retinoblastoma (usually with a solitary tumor) MECHANISMS FOR INACTIVATION OF 2ND ALLELE Rbmut Rb Heterozygous Tissue Chromosome Loss Rbmut Hemizygous Mutant Mitotic Recombination Chromosome Duplication Second Mutation Rbmut Rbmut Homozygous Mutant 71 MECHANISMS FOR INACTIVATION OF 2ND ALLELE - Mitotic recombination can lead to LOH of Rb gene - Genetic material is exchanged between two homologous chromosomes through the process of genetic crossing over (G2 or M phase) - Homo- or heterozygous daughter cells • • • • Gene conversion DNA polymerases initially begin to use the strand on one chromosome as a template for synthesis of daughter strand May continue replication by jumping to homologous chromosome and jump back A mutant TSG, like mutant allele of Rb, may be transmitted from one chromosome to its homolog, replacing the wild type Loss of heterozygosity - The Rb gene often undergoes LOH in tumors - Study of chromosome metaphase spreads - Second gene lying in 13q14 chromosomal region encodes enzyme esterase D - LOH affects whole region - Isoforms seperated by gel electrophoresis Loss of heterozygosity - The loss of genes localized to the region of LOH could result in haploinsufficiency or unmasking of the functional expression of a recessive or deficient allele. 72 Approaches to identify TSGs • • Cytogenetic studies to identify constitutional chromosomal alterations in cancer patients; deletion of specific chromosome or specific region: difficult due to small portion of cancer cells – Familial retinoblastoma; Ch13q14 in blood sample – Wilms tumor; 11q13 – adenomatous intestinal polyps; 5q Linkage analysis; trace the genetic markers from implicated chromosomal region co-segregated with the inheritance of the disease phenotypes; pinpoint the location of the TSG – • • LOH studies – Retinoblastoma > decreased levels of esterase D Positional cloning of the TSGs Monochromosome transfer Chromosome transfer Transfer of Human Chromosome 11 Suppression G401 Wilms Tumor Cells Transfer of Human Chromosome 13 Transfer of Human Chromosome X Tumorigenic Tumorigenic Chromosome transfer Transfer of Human Chromosome 11 Suppression G401 Wilms Tumor Cells Transfer of Chromosome 11del(p13) Transfer of Chromosome 11del(p15.5-p14.1) Suppression Tumorigenic 73 TSG inactivation by promoter methylation • • • • • Methylation of cytosine groups located in a position that is 5” to guanosines, in the sequence CpG > can alter DNA covalently In vicinity of promoter can repress transcription of gene Histone deacetylase enzyme complexes bind to Me-CpG, remove acetate groups and convert chromatin to non-transcriptive state Newly synthesized daughter strands are methylated by maintenance methylases > methylation state is heritable DNA methylation does not altered nucleotide sequence: epigenetic Methylation-specific PCR (MSP) to gauge methylation status of promoters - Bisulfite treatment causes conversion of cytosin to uracil - Methyl-cytosine is resistant - Primers will anneal to U-containing DNA recognize it as T - Non-methylated primers will fail to properly anneal to CpG that was methylated - Amplification of DNA fragments whose presence indicates methylation or absence thereof U behaves like T Methylation of the RASSF1A promoter • • • • • • • • • • Bisulfite sequencing technique to determine methylation of the CpG island in which the promoter of the RASSF1A tumor suppressor gene is embedded Each circle indicates the site of a distinct CpG dinucleotide Location of CpGs in promoter is indicated by map Blue circles indicate that CpG was methylated Open circle are unmethylated Analysis of 5 DNA samples Almost all CpG sites in RASSF1A CpG island are methylated Adjacent tissue contains some cells that are methylated No methylation in control tissue TSG suppression by hypermethylation 74 Methylation of the p16INK4A promoter • • • In-situ hybridization to determine methylation status of DNA Low and high grade cervical intraepithelial squamous lesions with varying degree of methylation Normal surface epithelium (arrow) is non-methylated Methylation of promoters of multiple TSG within tumor cell genomes INACTIVATION OF TUMOR SUPPRESSOR GENES Loss of Protein Function Small Sequence Mutations Missense mutants result in defective protein Insertion mutations result in defective protein Chromosomal Alterations Gene deletion results in loss of expression Gene rearrangement results in defective protein Epigenetic Regulation Overexpression of negative regulator inhibits protein function 75 TUMOR SUPPRESSOR GENES APC - Familial Adenomatous Polyposis (FAP) BRCA1 - Breast and Ovarian Carcinoma BRCA2 - Breast Carcinoma p53 - Breast, Colon, and Lung Carcinomas, (and others) Rb1 - Retinoblastoma, Osteosarcoma, Breast, Bladder, and Lung Carcinomas NORMAL FUNCTIONS OF TUMOR SUPPRESSOR GENES Regulation cell cycle progression Cellular differentiation Cell adhesion and cell structure Cell signaling Regulation of gene expression DNA replication and repair And others? NF1 (Neurofibromatosis type 1) • A common autosomal dominant disoder(1/3,500) • Also develop glioblastoma, pheochromocytoma, and myelogenous leukemia • Show Café au lait spots: hypergimentation, Lisch nodules, distinctive boney lesions etc • Chromsome 17q11.2 • Genetic behavior parallels Rb1 > LOH • Encodes neurofibromin • Functions as GTPase activating protein (GAP) 76 NF1 acts as an negative regulator of Ras signaling • Function as Ras-GAP protein • After growth factor stimuli, NF1 rapidly is degraded, but NF1 levels return back to normal to shut down further the Ras-signaling: Negative feedback • Cells lacking NF1 function have elevated levels of activated Ras • NF1+/- cells have elevated levels of Ras-GTP > substantially increased levels of Ras-signaling • Not both alleles have to be lost • “Haploinsufficiency” : p27, smad4, Pten etc, but not Rb, p53 Haploinsufficiency • • • • • • • Definition: a single wild type allele is unable to produce enough transcription product for normal cellular function A single mutant allele of a tumor suppressor poses an intermediate risk for cancer Not all mutant alleles are null recessives (alleles incapable of producing functional product) Hypomorphic recessive mutations: reduced but not absent functionality Dominant negative mutations: nonfunctional product that directly interferes with the function of the normal protein Pleiotrophy: functioning of one gene in multiple cellular processes. Some functions maybe more dosage dependant than others Extragenic modifiers: can both enhance and suppress the ability of tumor suppressors to prevent cancer Classic tumor suppressor genetics inherited cancer sporadic cancer Haploinsufficient tumor suppressor genetics Von Hippel - Lindau diseases: pVHL modulates the hypoxic response • • hypoxia inducible transcription factor-1 (HIF-1) is synthesized at a high rate under normoxia and immediately degraded by the actions of pVHL: proline hydroxylase oxydizes 2 prolin residues of HIF-1, which enables pVHL binding and ubiquitylation of HIF-1 under hypoxic conditions HIF-1 escapes ubiquitylation and activates VGEF 77 Cell cycle clock is the master governor to decide proliferation or not • Molecular circuitry operating in the cell nucleus that processes and integrates a variety of afferent signals originating from outside and inside the cell and decides whether or not the cell should enter into the active cell cycle or retreat into a nonproliferating state. • If cell proliferates, circuitry proceeds to program the complex sequence of biochemical changes that enable cell to divide into two daughter cells. Cell cycle regulation is based on cyclically activated serine/threonine protein kinases Changing levels/availability of cyclins > activate catalytic function of CDKs D type cyclins convey information about extracellular mitogenic signals Other cyclins operate on preordained schedule (move cycle only forward) Checkpoint controls ensure correct progression through cell cycle Cyclin-CDK complexes are also regulated by CDK inhibitors (p15, 16, 18, 19, 21, 27, 57) 78 Control of cell cycle by mitogenic signals • TGF-β induces p15INK4A (blocks CDK4/6) and weakly p21Cip1 expression • Akt/PKB phosphorylates p21Cip1 (nucleus) and p27Kip1 (cytoplasm) • phosph. p21Cip1 goes to cytoplasm, can’t inhibit cyclin-CDK complexes • phosph. p27Kip1 can’t go to nucleus to inhibit cyclin-CDK complexes • without active CDK4/6 cell can’t advance to R point, cell blocked in G1 • other cyclin-CDK complexes are active through remainder of cell cycle Inhibitory signal Stimulatory signal DNA damage p15INK4A Suppression of p27Kip1 function by Akt/PKB • various breast cancers, like breast carcinoma, show different p27Kip1 locations that reflect activation state of kinases Akt/PKB • phosphorylated p27Kip1 can’t go to nucleus to inhibits cyclin-CDK • cytoplasmic localization of p27Kip1 associated with decreased survival N = nuclear N+C = nuclear + cytoplasmic Cell cycle phosphorylation of Rb is the determinant of R point • Phosphorylation of Rb is closely coordinated with cell cycle advance • Cyclin D-CDK4/6 complex hypophosphorylates Rb • CyclinE-CDK2 complex hyperphosphorylates Rb (Active Rb) (Inactive Rb) 79 E2F transcription factor enables Rb checkpoint • E2F/DP1/RB complex represses gene transcription • hyperphosphorylation of Rb at R point activates E2F transcriptional activity • E2F is inactivated and degraded in S phase RESTING CELL Passage through the G1 restriction point Activation of E2F transcriptional activity Transcription of cell cycle genes (Cyclins A, E, CDK1) DNA replication genes Irreversibility of cell cycle progression • cyclin E-CDK2 complex drives Rb hyperphosphorylation > release of E2F from Rb control > synthesis of cyclin E: positive feedback loop • activation of small number of cyclin E-CDK2 > phosphorylation of p27Kip1 > marked for ubiquilation and degradation > liberation of more cyclin E-CDK2 Counterbalancing controls on cyclin D1 levels • mitogenic signals influencing cyclin D1: • 3 Ras signaling pathways: • AP-1 transcription factor (Fos/Jun) acts on cyclin D1 promoter > × • activates PI3 kinase and thus Akt/PKB > inhibits actions of GSK-3β > this spares β-catenin from phosphorylation, ubiquilation and degradation > β-cat partners with Tcf to induce cyclin D1 • activates Erk > opposite effect > phosphorylation of cyclin D1 > marked for ubiqilation and degradation • growth factors > growth factor receptors > Ras > cyclin D1 and E > inactivation of pRb > activation of E2F > S-phase entrance G1-S progression 80 Myc oncoprotein deregulates control of cell cycle progression • Myc modulates a number of positive and negative regulators of cell cycle advance • the heterodimer Myc-Max induces growthpromoting proteins cyclin D2 and CDK4 > hypophosphorylation of pRb > advance through G1 • Myc-Max increases expression of Cul1, which degrades the p27Kip1 CDK inhibitor liberating cyclin E-CDK2 complexes from inhibition • by association with second transcription factor, Miz-1, Myc can also act as transcription repressor: - represses expression of p15INK4B and p21Cip1 (CDK inhibitors that shut down CDK4/6 and CDK2), counteracts TGF-β • Myc can induce expression of genes encoding E2F 1, 2 and 3 > Ø pRb How TGF-β blocks the cell cycle progression • TGF-β “last word” in determining whether or not a cell proliferates • TGF-β through receptor phopshorylates Smad proteins, such as Smad 3 • Smad 3 and 4 form heterodimer • migrate to the nucleus • team up with Miz-1 • induce expression of p15INK4B and weakly p21Cip1 (CDK inhibitors that shut down CDK4/6 and CDK2) • Myc-Miz-1 repress expression of these CDK inhibitors • this action can be preemptively blocked by TGF-β by dispatching Smad 3 to form complex with E2F 4 or 5 plus p107 (cousin to pRb) > represses myc gene Cancer cell that have escaped growth-inhibitory influence of TGF-β often have pRb eliminated from this regulatory circuitry Rb also controls cell differentiation • The control of cell cycle differentiation is coupled to the regulation of cell cycle progression • Hypophosphorylated Rb is needed to halt proliferation of cells and facilitate differentiation • Dephosphorylation of Rb and blockage of cell cycle is a "differentiation decision" • Myoblasts only differentiate after proliferation stops (e.g. removal of growth factors) • bHLH transcription factor MyoD forms heterodimers with E12 and E47 to orchestrate myocyte differentiation • Id2 binds to MyoD and prevents differentiation • Expression of Id2 can be induced by Myc • In normal cells Id2 is sequestered by pRb • Overexpression of Id2 in neuroblastomas 81 Perturbation of the R point transition in tumors favor advance block advance The discovery of p53 • • • SV40 transformed tumor cells stained in the nucleus for SV40 large T antigen Immunoprecipitation of 94 kD protein only from virus infected 3T3 cells, also a 53 to 54 kD protein was detected in transformed 3T3 cells and both F9 cells LTA tightly bound to novel protein, now called p53, of cellular origin Wildtype p53 with ras: no tumor With mutated p53: tumor - few tumors with complete deletion - robust tumor with point mutation 3T3 Balb/c mouse fibroblasts F9 mouse embryonal carcinoma cells T = SV40 LTA N = negative Mutant versions of p53 interfere with normal p53 function Tumor-bearing Mice p53 +/+ p53 +/- 1% at 18 months 2% at 9 months Disruption of embryonic development p53 -/- 75% at 6 months No effect on embryonic development • p53 does not operate to transduce proliferative and anti-proliferative signals that continuously impinge on cells and regulate cell proliferation • p53 seems to specialize in preventing appearance of abnormal cells 82 p53 TUMOR SUPPRESSOR FUNCTION Interaction between the p53 protein and the DNA molecule is required for tumor suppressor function Amino acid residues in the p53 protein that contact the DNA molecule are frequently mutated Mechanisms of p53 dominantnegative mutations • p53 normally functions as a homotetrameric transcription factor • in cells bearing a mutant p53 allele that encodes an altered protein the structurally altered protein may retain its ability to form tetramers, but loose its ability to exert normal p53 function • a single mutant protein subunit may compromise tetramer function 15/16 tetramers with altered function Nature of p53 mutations • • • • point mutations are most common in p53 > dominant negative effect striking contrast to mutations in other tumor suppressor genes such as APC or “caretaker” genes involved in maintenance of the genome (ATM, BRCA) frame-shift mutations or nonsense codons in the majority of cases disrupt protein structure and create truncated, often defective proteins 83 Frequency of mutant p53 alleles in human tumor genomes p53 responds to various stressful signals Induction following DNA damage p53 mediated apoptosis • p53 uses multiple signaling pathways to activate apoptotic program • induces expression of genes encoding: • Fas receptor • Insulin like growth factor (IGF) F-binding protein-3 (IGFBP-3) • Bax • display of Fas receptor sensitizes cell to FasL induced apoptosis • release of IGFBP-3 from cell causes binding of pro-survival IGF1 and 2 • Bcl-2 related Bax is pro-apoptotic and causes release of cytochrome c p53 84 Control of p53 levels by Mdm2 (mouse double minutes) • following signaling p53 tetramer binds to the promoters of target genes including mdm2 • increase in Mdm2 protein • Mdm2 binds to p53 subunits • blocks functional ability of p53 • triggers ubiquitilation and after transport to cytoplasm degradation in proteasomes • negative feedback loop • phosphorylation of p53 blocks ability of Mdm2 to bind to p53 • by kinases ATM, Chk1 and 2 which become activated by DNA damage Consequences of p53 mutations • Li Fraumeni syndrome (familial cancer) • susceptibility to a wide variety of cancers due to mutations in p53 • green: breast cancer red: sarcoma • blue: lung cancer brown: Wilms tumor • yellow: glioblastoma purple: leukemia • orange: pancreatic carcinoma Cellular response to irradition 85 EVADING APOPTOSIS Differentiation Stimulated Inhibited Proliferation Cell Population Apoptosis Inhibited Stimulated Rates of cell proliferation and cell death in a homeostatic tissue are roughly equal. Perturbations in this homeostasis (increased proliferation or decreased death) can result in excessive tissue growth and ultimately may evolve into a tumor Tumorigenic Stress Both extracellular and intracellular stresses can induce tumorigenic changes in normal cells. Cells then attempt to avoid transformation by undergoing either cell-cycle arrest (time for the repair of DNA damage) or cell death. Several pathways, which involve some of the same signaling mediators, exist to prevent transformation, including those that lead to apoptosis, mitotic catastrophe, autophagy and necrosis. Anti-apoptotic strategies • decrease of pro-apoptotic protein function (labeled blue) • increase of anit-apoptotic protein function (labeled red) • commonly employed mechanisms by cancer cells to evade apoptosis: - inactivation of the p53 pathway - inactivation of Rb function - shutting down ARF - deregulation of bcl-2 like proteins - hyperactivation of the PI3K > Akt/PKB pathway - inactivation of PTEN - activation of NF-κB expression - expression of FLIP protein 86 Cellular failsafe mechanisms • aberrantly activated oncogenes induce failsafe mechanisms in the cell to prevent unregulated proliferation • cells exit cell cycle via CDK4 inhibitor INK4A and Mdm2 inhibitor ARF • activated Ras or Myc induce p53 and Rb anti-proliferative activities • oncogenic RAS leads to upregulation of INK4A, blocks cyclin-D/CDK4-mediated hyperphosphorylation of RB, provokes cellular senescence • activated MYC induces ARF, blocks p53 inhibitor Mdm2, activates p53 • tumorigenesis relies on the cancellation of these failsafe mechanisms Control of apoptosis by ARF • ARF (p14ARF) can associate with Mdm2 • drags Mdm2 into nucleolus > can’t attack p53 there > ×ARF = ×p53 • numerous oncogenic signals (e.g. Myc, Ras) favor apoptosis and induce E2F activity • × ARF expression • inactivation of one allele of ARF > rapid tumor development in mice with Eµ-myc transgene Actions of the anti-apoptotic Bcl-2 gene • clones of myc and bcl-2 were placed under control of promoter IgG > expression in B cell lineage • induced in germ lines of mice • mice carrying various transgene • no mortality in bcl-2 transgenes, greatly increased mortality in myc transgenes • acceleration of lymphomagensis in mice with myc and bcl-2 transgene • bcl-2 prolongs the life of lymphocytes that would otherwise undergo apoptosis 87 Actions of the anti-apoptotic Bcl-2 gene • chromosomal breakpoint in human follicular B-cell lymphomas • translocation generates a fusion between Ig gene on chromosome 14 and the bcl-2 gene on chromosome 18 • high levels of expression of bcl-2 mRNA in mature B-cells • increased Bcl-2 protein levels impedes apoptosis in B-cells, normally a high turnover rate • end result: follicular lymphoma • relatively indolent cancer (small resting B-cells) • often progresses to malignant high grade lymphoma Exploitation of apoptosis by cancer therapy treatments • Eµ-Myc mouse tumors treated with DNA damaging agents • trigger a p53-mediated apoptotic response, tumor burden is lowered • apoptotic response is prevented by Bcl-2 overexpression Rapamycin and the mTOR circuit • mammalian target of rapamycin • signals: nutrients, oxygen, ATP, mitogenic signals • responses: cell proliferation, protein synthesis, angiogenesis, anti-apoptotic, ribosome biogenesis, cell motility • exists in alternative complexes: • Rictor > activity of Akt/PKB • Raptor > activates protein synthesis • rapamycin blocks mTOR-Raptor • also shuts down mTOR-Rictor • primary effect is inhibition of Akt/PKB signaling (carcinomas with loss of PTEN) 88 Mitotic catastrophe as a failsafe mechanism • type of cell death caused by aberrant mitosis: characterized by multinucleate giant cells that contain uncondensed chromosomes • integrity of M-phase progression is monitored by: • detection of DNA damage (G2/M arrest) • detection of mitotic-spindle malformations • detection of incorrect positioning of the spindle • defects in genes required for MC can contribute to tumorigenesis Mitotic catastrophe as a failsafe mechanism • numerous kinases involved in mitotic regulation: polo-like kinase 1 (PLK1), aurora kinase family (aurora-A and -B), regulator of spindle checkpoint called BUB-related kinase (BUBR) • response to genotoxic stress: ataxia teleangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) are activated and activate the checkpoint kinases CHK1 and 2 > phosphorylate and inactivate CDC25 > translocated into the cytosol > blocks CDK1 activation > G2 arrest Consequences of DNA damage • results in immediate arrest in the interphase of the cell cycle or can cause a transient entry into the prophase (reversible chromatin condensation) with a prolonged 'antephase‘ • failure to activate the checkpoints, causes entry into mitosis and death during the metaphase • in case of mitotic slippage and consequent mitotic failure, generation of tetraploid cells that undergo apoptosis or generate aneuploid cells 89 Autophagy and beclin 1 • adaptation to nutrient limitation by catabolizing intracellular components to ensure survival, also targeted elimination of proteins and organelles • can lead to cell death under prolonged nutrient starvation • Beclin-1 is a key regulator of the autophagy process • activated by nutrient limitation or damaged or excess organelles • wortmannin and 3-methyladenine inhibit beclin-1 > inhibit autophagy • 40-75% of breast or ovarian carcinomas have deletions of beclin-1 gene Autophagosome Autolysosome 2) Nucleation/ 3) Membrane Assembly elongation 4) Fusion 5) Degradation Senescence and Immortality Replicative senescence a non-growing state of cells in which they exhibit distinctive cell phenotypes and remain viable for extended periods of time, but are unable to proliferate again loss of proliferative capacity with age Immortality trait of a cell or population of cells to proliferate indefinitely Telomeres • telomeres are TTAGGG repetitive sequences that cap the end of eukaryotic chromosomes • protect the ends of chromosomes from degradation and from end-to-end fusion with other chromosomes • a core of telomere binding proteins termed the sheltering complex, serve to protect telomeric ends • critical shortening or uncapping of telomere binding proteins results in telomere dysfunction • dysfunctional telomeres activate a DNA damage response • in cells with functional p53 pathway, this initiates cell crisis and apoptotic programs to inhibit tumorigenesis • in cells with mutant p53, dysfunctional telomeres promote genomic instability and progression to cancer 90 Replicative lifespan • normal cells: mortal, no telomerase activity, telomere shortening with divisions • cancer cells: immortal, express telomerase, stable telomere lengths • normal cells have limited replicative lifespan, enter irreversible growth arrest, replicative senescence (M1) after extended passage • introduction of viral oncoproteins (SV40) before M1 allows continued proliferation with further shortening of telomeres > second proliferative barrier, crisis (M2) • crisis: state arising when cells lose telomeres of adequate length, resulting in end-to-end fusion of chromosomes, karyotypic chaos, and widespread apoptosis • activation of telomerase through introduction of the catalytic subunit of telomerase, TERT, allows cells to avoid replicative senescence Breakage-Fusion-Bridge (BFB) cycles • occurs when telomeres are too short to protect chromosomal DNA (in G2 phase, telomeres were eroded in duplication in S phase) • during normal mitosis 2 sister chromatides are pulled apart to opposite centrosomes • centromeres in fused chromosome will also be pulled apart • dicentric chromosomes are unable to separate • create bridge between poles of mitotic spindle • break at weak point • new fusions in next cell cycle • repeat of earlier events • BFB cycles create karyotypic chaos Telomere structure • TTAGGG repetitive sequences that terminate in a 3’ single-stranded overhang (ss) • ss overhang can invade the double stranded region of the telomere to form a protective telomere loop at the invasion site (t loop) • telomeric DNA is complexed by the sixprotein-sheltering complex of telomericrepeat binding factors 1 and 2 (TRF1 and 2), RAP1, TRF1-interacting nuclear factor (TIN2), TPP1, POT1 • TPP1-POT1 heterodimer regulates access of telomerase to the telomeric substrate • telomeres transiently interact with a host of other factors, many of which are involved in DNA damage response G-rich strand 91 End replication problem • during DNA replication the parental DNA double-helix is unwound by helicase • new lagging strand synthesis by short RNA primers that lay down intervals • new leading strand synthesis can proceed continuously (5’ to 3’ direction) • also initiated by an RNA primer that sits on 3’ end of parental strand • loss of primer binding site, “under-replication” of parental strand Telomere dysfunction • progressive shortening of telomeres initiates DNA damage response • activation of ataxia telangiectasia mutated (ATM) and ataxia-telangiectasia and -Rad3related (ATR) and kinases CHK1 and 2 • uncapping of TRF2 engages ATM • uncapping of POT1 engages ATR • phosphorylation of p53 > regulation of senescence and apoptosis • transcription of p21 induces a senescentlike growth arrest, cannot be reversed by physiological mitogens, but is reversible upon inactivation of p53 • oncogenes and other types of stress induce p16, which activates pRB • pRB-mediated senescence arrest cannot be reversed by inactivating p53, pRB, or both Telomerase • the telomerase holoenzyme is composed of at least 2 essential subunits • hTERT (human telomerase reverse transcriptase) catalytic subunit and the hTR RNA subunit (telomeraseassociated RNA molecule) • the holoenzyme attaches to the 3’ end of the G-rich strand overhang • reverse transcription of sequences present in hTR subunit • extends the G-rich strand by 6 nucleotides • process is repeated hundreds of times 92 LIMITLESS REPLICATIVE POTENTIAL • ss overhang degraded in cells entering replicative senescence • blunted DNA emits damage signal • activation of p53 • p16INK4A which is activated by physiologic stress • together they induce senescent growth state • telemorase expression, catalytic subunit hTERT) can prevent cells from entering into replicative senescence by reversing loss of G-rich overhang • cells in culture with ectopically expressed telomerase continue to grow, proliferate indefinetely LIMITLESS REPLICATIVE POTENTIAL • some cells maintain telomeric DNA without actions of telomerase • alternative lengthening of telomeres (ALT) used by some cancer cells • DNA polymerases use more than one template during replication • one telomere extends its overhang, which displaces a strand of same polarity in the telomere of another chromosome (3’ end anneals) • conventional polymerases extend strand using complementary strand as template • the recently elongated strand disengages and can be converted into double strand • process is repeated multiple times copy choice mechanism BFB cycle promotes carcinoma formation • inactivation of p53 during early stages of tumor progression (favors escape from apoptosis due to oncogene activation etc.) • as tumor progression proceeds, the telomeric DNA of pre-malignant cells will erode beyond chromosomal protection • BFB cycles will ensue • leads to increased chromosomal rearrangement and amplification and deletion of chromosomal segments close to breakpoints • cells not eliminated by p53 (see above) • variants will emerge that can regenerate telomeres and thereby stabilize their karyotype, cells that can grow rapidly • activation of ALT mechanism, derepression of hTERT expression 93 TISSUE INVASION AND METASTASIS Cancers grow by progressive infiltration, invasion, destruction, and penetration of the surrounding tissue SPREAD OF NEOPLASMS Local invasion Implantation Hematogenous Lymphatics Risk of metastasis in correlation to primary breast carcinoma size SPREAD OF NEOPLASMS Direct invasion of normal lung by lung SCC (right), and tumor spread through bronchioles (left) 94 SPREAD OF NEOPLASMS Infiltration and dissection along tissue planes Requires a lack of cohesiveness between cells Enzymatic degradation of extracellular matrix Extension of pseudopodia between cells Mesothelioma on the pleural surface of the lung SPREAD OF NEOPLASMS Lymphatic spread and lymph node involvement by metastatic tumors will reflect the natural lymphatic drainage of the tissue site of the primary tumor. Mammary carcinoma spreading through a lymph node (left), and mammary carcinoma in a dilated lymphatic vessel of the lung (right) SPREAD OF NEOPLASMS Hematogenous spread occurs principally through the venous circulation. Consequently, the liver and lungs are the most frequently involved secondary sites. Invasive mammary carcinoma metastatic to liver. 95 ORGAN PREFERENCES FOR METASTASES • Hemodynamic and Anatomic factors • Filtering organs often sites of metastasis: • Extensive capillary network of the lung and liver • Regional lymph nodes • Sites of endothelial damage: • Interaction of neoplastic cells with platelets and fibrin • Tissues with large volume of blood flow: • Brain and Kidney ORGAN PREFERENCES FOR METASTASES • Intrinsic factors of neoplastic cells • Neoplastic cells need hospitable environment to colonize: “seed and soil theory” (contralateral mets?) • Neoplastic cells may possess tissue-specific cell membrane proteins which interact with endothelial cell proteins to facilitate metastasis: vascular ZIP code • Metastatic sites may be pre-programmed and not random: “The coconut theory” ORGAN PREFERENCES FOR METASTASES 96 Metastases to bone • requires subversion of osteoclasts and osteoblasts • osteolytic or osteoblastic lesions • receptor activator of NF-κB ligand (RANKL) is important mediator of osteoblastic differentiation • RANK binding on osteoclasts precursors to RANKL on osteoblasts results in differentiation to functional osteoclasts: osteolysis • osteoprotegerin (OPG) can prevent RANKL binding • OPG – RANKL balance determines the net rate of bone growth/loss Osteolytic bone metastases • release by tumor cells of PTHrP > acts on osteoblasts > increase RANKL release, blocks OPG > maturation of osteoclasts > bone lysis > exposure of bone ECM > release of TGF-β1, Ca2+, IGF-1, PDGF, FGFs, BMPs > tumor cell survival and proliferation • TGF-β1 initiates positive feedback loop > release of more PTHrP • “vicious cycle” of osteolytic mets • breast cancer, anal sac carcinoma • Prostate cancer causes osteoblastic metastases through release of endothelin 1 growth factor Metastatic tropism and gene expression IL11 (interleukin11), OPN (ostopontin), CTGF (connective tissue growth factor), CXCR4 (chemokine receptor 4), MMP1 (matrix metallo proteinase 1) Original tumor Metastatic subclone • 33 single cells from mammary carcinoma clonally expanded • analyzed mRNA pattern • visualization of mets with luciferase gene • 5 genes analyzed since they are overexpressed in bone metastases and promote osteolysis • bone metastases: all 5 genes are high (clone 2), lung metastases: all 5 genes are low (clone 3), no metastases: no gene expressed (clone 26) GAPDH (glyceraldehyde 3 phosphatase dehydrogenase) 97 SPREAD OF NEOPLASMS The invasion-metastatic cascade Most malignant cells released from tumors die in circulation Detachment of tumor cells from each other - E-cadherins mediate adhesions - linked to cytoskeleton by catenins Attachment to matrix components - receptor-mediated - to laminin and fibronectin - express higher amount of integrins - produce integrins that are not present in the normal tissue Degradation of ECM - chemical alteration of connective tissue matrix - 3 classes of proteases: serine, cysteine, and matrix metalloproteinases (MMPs) - MMPs promote angiogenesis, tumor growth, tumor cell motility Patterns of local invasion • individual cells leave primary tumor one by one (“Indian file”) through channels in stroma • phalanx (“well organized cohort”) of cells invades nearby stroma, more typical behavior E-cadherin metastatic melanoma cells collagen β1 integrins 98 Steps leading to extravasation cancer cells are physically trapped in capillaries > attach to platelets > form microthrombus > degranulate, release growth factors, proteases etc. > push aside endothelial cell on one side of wall (contact to basement membrane) > microthrombus is dissolved by proteases > proliferation of cancer cells in lumen > break through basement membrane Tumor progression Darwinian evolution and clonal expansion of advantages genotypes and thus phenotypes Selection, outgrowth - more growth autonomous - ignore death and senescence signals - escape immune surveillance - trigger angiogenesis - invasion, metastasis Nonviable (antigenic, apoptotic, etc.) The evolution of colonizing ability Initially formed genetically heterogenous primary tumor cell population seeds equally heterogeneous micrometastases Surgically removed primary tumor leaves behind minimal residual disease Micrometastasis aquires ability to colonize, i. e. grow into macrometastasis New source for micrometastases that are genetically very similar to another Grow fast new macrometastases since the have ability to colonize 99 Epithelial-mesenchymal transition • shedding of epithelial characteristics to acquire motility and invasiveness • in normal epithelium Ecadherin is tethered to actin cytoskeleton via αand β-catenin • loss of E-cadherin liberates β-catenin which migrates to the nucleus • associated with Tcf/Lef transcription factors and key intermediary in Wnt pathway • initiates EMT program β-catenin and the biology of colonic crypts • • • • • Stem cells in crypts: high levels of β-catenin, signaled by stromal Wnt β-catenin associates with Tcf/Lef transcription factors > increased proliferation > cells migrate to lumen > decrease in Wnt and increase in Apc > increased degradation of β-catenin > cessation of proliferation and apoptosis Apc (adenomatosis polyposis gene) negatively controls β-catenin levels Apc brings together glycogen synthase kinase 3β and β-catenin > phosphorylation of β-catenin > degradation by ubiquitin-proteasome pathway Apc inactivation leads to high levels of β-catenin Cadherin shift and invasiveness • shift from E- to N-cadherin in melanomas enables migratory behavior • shift facilitates invasion into stroma since melanocytes can dissociate themselves from neighboring keratinocytes without E-cadherin • N-cadherin allows homotypic interactions with various cell types 100 Control of EMT by TGF-β • Ras transformed mammary tumor cells express E-cadherin, but when grown in presence of TGF-β they undergo EMT and express vimentin • Ras downstream effectors Raf and PI3K induce TGF-β secretion • tumor cells express αVβ6 integrin while TGF-β is expressed by stroma • αVβ6 activates latent TGF-β in stromal cells > positive feedback loop Role of NF-κB in EMT • Ras transformed mammary tumor cells express E-cadherin, but TGF-β suppresses E-cadherin and induces vimentin expression • NF-κB signaling is suppressed > TGF-β fails to suppress E-cadherin • NF-κB is necessary for EMT and constitutive activation of NF-κB will induce EMT in cancer cells Macrophages stimulate invasive behavior • breast cancer cells recruit large numbers of tumor associated macrophages (TAM) • TAMs are found in close proximity to microvessels and are the major source of EGF • EGF stimulates cancer cells to release colony stimulating factor (CSF-1) • as a consequence CSF-1 stimulates EGF production: positive feedback loop • TAM derived TNF-α further contributes to EMT • TAMs also release MMPs 101 Macrophage contribution to tumorigenesis • carcinoma cells recruit circulating monocytes into tumor where they differentiate into macrophages (TAM) • recruited by chemotactic factors: • monocyte chemotactic protein (MCP-1) • colony stimulating factor (CSF-1) • platelet derived growth factor (PDGF) • TAMs secret EGF > cancer proliferation • hypoxic areas attract TAMs > secret VGEF > reduces hypoxia by bringing in endothelial cells > angiogenesis • TAMs secret MMP-9 (gelatinase B) and degrade ECM and cleave other proteins • Cleave insulin like growth factor binding proteins (IGFBP) > liberates IGF > survival signals to cancer cells Invasive behavior induced by HGF • hepatocyte growth factor (scatter factor: SF) is produced by a wide variety of stromal cells • Met is the cognate receptor expressed on epithelial cells • epithelial cells stimulated with HGF become motile and scatter through growth medium • in collagen gels HGF stimulated cells invade into surrounding gel Signals that trigger EMT • TNF-α, EGF, HGF, FGF induce Ras pathway that stimulates TGF-β and blocks apoptotic effect of TGF-β through PI3K • NF-κB, TGF-β and β– catenin induce EMT in cancer cells: motility and invasiveness • not only genotype dictates cancer cell phenotype, but also heterotypic interactions (depend on normal cells) and microenvironment 102 Embryonic EMT signaling • striking similarities in EMT signaling between early embryogenesis and tumorigenesis • further support for the notion that the EMT program expressed by invasive carcinoma cells represents a reactivation of latent cell-biological programs, many of which are active in early mammalian embryonic development Extracellular proteases in invasiveness • proteases are key effectors of EMT • mainly matrix metallo-proteinases (MMPs) • produced by macrophages, mast cells, fibroblasts and other stromal cells • some cancer cells may have induced synthesis of MMPs, mainly MMP-2 and 9 • MMPs cleave collagens, laminin, tenascin, fibronectin and proteoglycans • synthesized as inactive zymogens and activated by proteinase cleavage • 187 MPs: only 28 are secreted MMPs, 6 are membrane-anchored (MT1-MMP) • MT1-MMP can only cleave proteins in close proximity to cell • can activate pro-enzymes like pro-MMP2 • activity can be confined: podosomes Urokinase plasminogen activator (uPA) • inactive pro-enzyme of uPA is released from stromal cells • binds to its cognate receptor, uPAR, which is displayed on epithelial cells • binding converts uPA into catalytic active protease that can convert plasminogen into active plasmin • plasmin functions as protease to cleave pro-enzyme forms of MMPs and latent form of TGF-β • active forms degrade ECM • there is also evidence that uPA can directly convert pro-MMPs 103 MMPs in tumorigenesis • proteases are mainly effectors of EMT, but can drive progression of cells through all stages of multi-steps tumorigenesis • in particular MMP-3 (stromelysin-1) • overexpression of MMP-3 in mammary glands of transgenic mice will lead to hyperplasia and eventually progress to malignancy Locomotion on solid surfaces • locomotion of cultured cells depends on coordination of a complex series of changes in cytoskeleton and breaking of focal contacts with growth substrate • cell organizes actin fibers to extend lamellipodia on edge • filopodia protrude from these • cell surface proteases degrade proteins that stand “in the way” • integrins establish new points of contacts at lamellipodia edge • actin and myosin II stress fibers contract cell on trailing edge to break focal contacts The circuitry mediating cell motility • motogenic growth factors EGF, HGF and PDGF stimulate circuitry • Rho family of small GTPases ( i.e. Rho, Rac, Cdc42) has central role in controlling actin cytoskeleton and focal adhesions • stimulation of Ras effector PI3K allows guanine nucleotide exchange factors (GEF) to attach to membrane and to activate Rho GTPases • Tiam1 (T cell lymphoma and metastasis gene) is stimulated by Ras and PIP3 • Tiam1 GEF polymerizes actin on leading edge of migrating cells 104 Metastasis suppressor genes • relatively little know, early stage of research • many genes encode growth factors, growth factor receptors and signal-transducing proteins that when introduced in epithelial cells encourage EMT, acquisition of motility and invasiveness • deregulated versions of these genes may drive metastasis and invasion • mechanisms of E-cadherin or TIMP seem obvious, others are unknown SUSTAINED ANGIOGENESIS • oxygen, amount of nutrients, ability to shed metabolic waste products require close proximity to vasculature • necrosis is the primary result of hypoxia in poorly vascularized areas • oxygen pressure drops to zero at a distance of more than 0.2 mm • tumor can not only depend on genetic blue print for growth, but has to rely on heterotopic interactions when designing the layout of its own vasculature supply • endothelial cells, pericytes and smooth muscle cells all play a critical role in tumor angiogenesis: VEGF, TGF-β, basic fibroblast growth factor (bFGF), IL-8, angiopoietin, angiogenin, PDGF Angio- and lymphangiogenesis • angiogenesis (the formation of new blood vessels from existing ones) is an important process in the growth of malignant tumors • an association between VEGF and angiogenesis, malignancy, and metastasis has been established • many pro- and anti-angiogenic cellular factors regulate angiogenesis • major steps of endothelial cells in angiogenesis: - breaking through of basal lamina that envelops existing blood vessels - migration towards source signal - proliferation - formation of tubes • blood and lymphatic network derive from common precursors in embryo • VGEF-A and -B stimulate vascular growth • VGEF-C and -D stimulate lymphatics • VGEFs and VGEF-Rs are homologous • no support by mural cells in lymphatics 105 Tripping the angiogenic switch • “angiogenic switch”: moment at which a tumor begins to overexpress pro-angiogenic factors such as VEGF • malignant tumors need to attract blood vessels to grow beyond 0.2 mm • transgenic Rip-Tag mouse model: - normal pancreatic islets have small number of capillaries for support - 50% of islets: hyperplasia (-0.2 mm) - remain in dynamic no growth state - islet cells express VGEF which is sequestered by ECM - 10% islets become angiogenic - recruit mast cells and macrophages - both release MMP-9 > activate VGEF - 3% malignant transformation Angiogenic switch initiates a complex process • angiogenesis involves stroma prior to breakdown of basement membrane • PIN (carcinoma in-situ) recruits accumulation of stromal cells through intact basement membrane • tumor invasiveness and angiogenesis are tightly coupled • microvessel density is commonly associated with malignancy • tumor needs to recruit endothelial precursor cells (EPC) from bone marrow, stimulated by VGEF and stroma-derived-factor-1 (SDF-1) that is released from myofibroblasts • EPC reach tumor through blood circulation and differentiate Prostate intraepithelial neoplasia (PIN) Angiogenesis inhibitors • angiogenesis is normally suppressed by physiologic inhibitors • suppression of hypoxia-inducedfactor-1 (HIF-1) central in healing • ECM components block angiogenesis: • thrombospondin-1 (Tsp-1) binds to CD36 on endothelial cells and halts proliferation • Tsp-1 also releases Fas ligand from endothelial cells > apoptosis • Fas receptor only on proliferating cells • Tsp-1 is induced by p53 and can be shut down by Ras oncogene • endostatin, endorepellin, troponin 1, tumstatin, arresten, TIMPs etc. • non-matrix components: IFN-α, IL-1, etc. 106 Balancing the angiogenic switch • diagram of major physiologic regulators that promote or inhibit angiogenesis • in reality not a binary decision, but switch has many gradations of development during tumorigenesis • highly complex systems are more vulnerable to disruption • at least a dozen factors involved in regulating vascular morphogenesis • complexity offers multiple targets for intervention • much easier to target angiogenesis since normal cells are targeted and not tumor cells that commonly become therapy resistant Therapeutic considerations • anti-VGEF-R drug effective on blocking angiogenic switch in hyperplastic islets • anti-PDGF-R-drug more effective late stage • VGEF-R only important in early stages of angiogenesis, but PDGF-R vital in recruiting pericytes to growing capillaries, protects them against anti-angiogenic signals anti- VGEF-R and PDGF-R combination therapy Therapeutic considerations Heterotopic interactions as target for therapy 107 THE ROLE OF THE STROMA Cross talk between the ECM and tumor cells cleavage of matrix components (type IV collagen), degradation of laminin by MMP-2 release angiogenic factors and proteolytic fragments that favor cancer cell motility ECM stores growth factors in inactive forms PDGF, TGFβ, and b-FGF affect the growth of tumor cells in a paracrine manner Stromal cells transmit oncogenic signals Stroma can drive genetic changes that promote carcinogenesis CARCINOGENESIS IS A MULTISTAGE PROCESS Initiation Promotion Conversion Progression Defects in Cellular Differentiation Defects in Growth Control Resistance to Cytotoxicity Insult Genetic Change Normal Cell Selective Clonal Expansion Genetic Change Genetic Change Initiated Preneoplastic Malignant Cell Lesion Tumor Genetic Change Clinical Cancer Advanced Clinical Cancer Activation of Proto-oncogenes Inactivation of Metastasis Inactivation of Tumor Suppressor Genes Suppressor Genes Colorectal cancer • a series ranging from single crypt lesions (aberrant crypt foci) through small benign tumors (adenomatous polyps) to malignant cancers • inherited factor : HNPCC (hereditary nonpolyposis colorectal cancer) and FAP( familial adenomatous polyposis) • activation of RAS oncogenes (50 % of colorectal cancers, c-Kis-RAS, N-RAS) and inactivation of TSGs on ch 5q (p53 gene), 17p (DCC, SAMD4/DPC4, SMAD2) • mutation of APC-tumor suppressor gene on ch 5q, increased β-catenin/Tcf-mediated transcription • several inherited predispositions can result from inheritance of a single defective gene – caretakers (DNA MMR gene in HNPCC), gatekeepers (APC), • definition of a model for colorectal tumor development – mutations of APC→K-RAS→18q suppressor and p53 • accumulation of genetic changes is facilitated by a chromosomal instability 108 Inherited predisposition 1. Presence of polyposis (FAP) • develop hundreds to thousands of adenomatous polyps during their lifetime • colorectal cancer develop at median age of about 40 • increased risk for thyroid, small intestine, stomach, and brain • GS (Gardner syndrome), CHRPE, Turcot syndrom are variants of FAP • germ-line mutations of the APC gene 2. Absence of polyposis (HNPCC) • develop at median age of about 42 years • defect in DNA MMR genes • at increased risk for uterus, ovary, brain and other cancers Other genetic Factors and environmental factors(diets) • associated with increased risk for colorectal cancer The APC gene • expression increases as cells migrate to top of crypt • β-catenin, γ-catenin, GSK-3β, AXIN family proteins, EB-1 and hDLG bind to the C-terminus of APC → all APC mutations result in loss of the C-terminus of APC → no interaction • mutation of APC : prevent its inhibition of β-catenin • dominant activating β-catenin mutations that render the protein insensitive to APC/GSK-3β-mediated degradation • increased β-catenin/Tcf mediated transcription results in expression of genes that promote cell growth or inhibit cell death (ex. c-MYC) • APC binds to tubulin and stabilize the microtubule, possibly involving the chromosomal stability THE PATHOGENESIS OF HUMAN COLORECTAL CANCER Normal Colonic Mucosa Hyperplasia Intermediate Adenoma Late Adenoma Carcinoma Metastasis 109 THE PATHOGENESIS OF HUMAN COLORECTAL CANCER CANCER STEM CELL THEORY • Stem cells as a source of neoplasms • Neoplasms don’t usually develop from completely differentiated cells that undergo de-differentiation • Cells within a tumor that have the capacity to initiate and sustain the tumor • These cells maintain their self-renewing capacity, have the ability to invade surrounding tissue and evade apoptosis “ONCOGENY AS PARTIALLY BLOCKED ONTOGENY” CANCER STEM CELL THEORY Normal stem cells Rare cells within organs with the ability to selfrenew and give rise to all types of cells within the organ to drive organogenesis Cancer stem cells Rare cells within tumors with the ability to selfrenew and give rise to the phenotypically diverse tumor cell population to drive tumorigenesis 110 Properties shared by normal stem cells and cancer stem cells Assymetric Division: • Self renewal - Tissue-specific normal stem cells must self-renew throughout the lifetime of the animal to maintain specific organs - Cancer stem cells undergo self-renewal to maintain tumor growth • Differentiation into phenotypically diverse mature cell types - Give rise to a heterogeneous population of cells that compose the organ or the tumor but lack the ability for unlimited proliferation (hierarchical arrangement of cells) • Regulated by similar pathways - Pathways that regulate self-renewal in normal stem cells are dys-regulated in cancer stem cells Two general models for cancer heterogeneity 1. All cancer cells are potential cancer stem cells but have a low probability of proliferation in clonogenic assays 2. Only a small definable subset of cancer cells are cancer stem cells that have the ability to proliferate indefinitely. Two general models for cancer heterogeneity Self renewal and differentiation are random. All cells have equal but low probability of extensive proliferation. Only cells with self renewal capacity can sustain tumor growth. Distinct classes of cells exist within a tumor. Only a small definable subset, the cancer stem cells can initiate tumor growth. 111 Pathways involved in self-renewal that are deregulated in cancer cells • Wnt, Shh, and Notch pathways contribute to the self-renewal of stem cells in a variety of organs • dysregulating these pathways contributes to oncogenesis • mutations of these pathways have been associated with a number of tumors: • colon carcinoma (Wnt), basal cell carcinoma (Shh), leukemia (Notch) Development of hematopoietic stem cells • subdivided into long-term and short-term selfrenewing HSCs and multipotent progenitors • give rise to CLPs (common lymphoid progenitors) and CMPs (common myeloid progenitors • CMPs/GMPs (granulocyte macrophage precursors) and CLPs can give rise to all known dendritic cells • MEP = megakaryocyte erythrocyte precursor Stem Cells CD34+ CD38- Multipotent Progenitors Oligolineage Progenitors CD34CD38+ Mature Cells CD20+ CD8+ CD8+ Differentiation Self Renewal CD34CD38- CD4+ CD4+ CD36+ CD35+ The importance of self-renewal in leukemic initiation and progression Self-renewal is a key property of both normal and leukemic stem cells. Fewer mutagenic changes are required to transform stem cells in which the self-renewal machinery is already active (a), as compared with committed progenitors in which self-renewal must be activated ectopically (b). In addition, self-renewing stem cells are long-lived; thus, there is an increased chance for genetic changes to accumulate in individual stem cells in comparison with more mature, short-lived progenitors. 112 Hematopoietic Cancer Stem Cells Acute myeloid leukemia (AML) – CD34+ CD38- Leukaemic Mouse Models: chronic myelomonocytic leukaemia (CMML) MRP8-BCL-2 acute myeloid leukaemia (AML) MRP8-BCL2Xlpr/lpr chronic myeloid leukaemia (CML)/Blast MRP8-PML-RARα acute promyelocytic leukaemia (APML)77 MRP8-BCRablXBCL-2 CANCER STEM CELL THEORY Clonal Genetic Model of Cancer Epigenetic Progenitor Model of Cancer tumor-progenitor genes = TPG gatekeeper mutation = GKM tumorsuppressor gene = TSG oncogene = ONC Therapeutic implications of Cancer Stem Cells • Most therapies fail to consider the difference in drug sensitivities of cancer stem cells compared to their non-tumorigenic progeny • Most therapies target rapidly proliferating nontumorigenic cells and spare the relatively quiescent cancer stem cells 113 Chronic inflammation and infection can increase cancer risk Inherited Disease Acquired Tumor Site Risk Hemochromatosis Liver Hereditary Pancreatitis Pancreas Ulcerative Colitis Colon Crohn’s Disease Colon 219 53 6 3 “18% of human cancers, i.e., 1.6 million per year, are related to infection.” - B. Stewart and P. Kleihues World Cancer Report, IARC Press, p. 57, 2003 Disease Tumor Site Viral Hepatitis B Hepatitis C Liver Liver Bacterial Helicobacter Pylori Gastric PID Ovary Parasitic S. hematobium S. japonicum Liver Fluke Risk 88 30 11 3 Urinary Bladder 2-14 Colon 2-6 Liver 14 Chemical/ Physical/Metabolic Acid reflux Esophagus Pancreatitis Pancreas Asbestos Lung pleural Obesity Multiple sites 50-100 17 >10 1.3-6.5 Contributions of free readicals to carcinogenesis • introduction of DNA Damage • activation of the protective p53 stress response pathway • mutation of cancer-related genes • activation of the NFκB pathway • activation of COX2 and the arachidonic acid cascade • activation of the hypoxic protective state by stabilization of HIF1α • promote clonal selection of cells resistant to the toxic effects of free radicals, e.g., p53 mutant cells Downstream effects of NF-kB 114 Mechanisms of activation of NF-kB Free Radicals Epithelial inflammation and tumor promotion • • • • • inflammatory stimuli, including chronic infection, result in recruitment of inflammatory cells into areas of inflammation TNF-α released by inflammatory and endothelial cells activation of NF-κB pathway production of more TNF-α, antiapoptotic protein (e.g. Bcl-XL), mitogenic proteins (e.g. Myc) also production of COX-2, which synthesizes multiple prostaglandines that induce different cancer phenotypes: loss of contact inhibition, increased proliferation, anchorage-independent growth Innate Immune Response Experimental pathway 12-O-tetradecanoylphorpbol-13-acatate) Stress Adaptive Immune Response Inflammation • Cytokines • Chemokines • Free Radicals • Prostaglandins • Growth Factors • MMPs Epithelium Adaptive Immune Response • Cytotoxic T cell Activity • Tumor Cell Lysis • Humoral Immune Response • T-Reg Cells Stroma • DNA Damage • Protein Modification • Proliferation • miRNA Expression • DNA and Histone Methylation • Angiogenesis CANCER Landscape Effects Metastasis 115 Molecular Based Diagnostic Tests in Canine Hematologic Malignancies • Detection of individual mutations in oncogenes: c-kit, p53 • Detection of chromosomal translocations, deletions and duplications: bcr-abl • Detection of clonality in lymphomas Response to Cytotoxic Drugs p53 Response to Cytotoxic Drugs Caspase 3 116 bcr-abl Translocations Producing a Fusion Protein • abl gene (Abelson murine leukemia virus), rapidly tumorigenic retrovirus, maps to chromosome 9q34 • Fused with sequences clustered at 22q11, called breakpoint cluster region = bcr (both are multidomain, multifunctional proteins) • Identified in 95% of Chronic myelogenous leukemia (CML) • Expression of bcr/abl gene driven by bcr gene promoter, produced transcript represents a chimera consisting of 5’ portion of bcr gene, and 3’ portion of cabl • bcr/abl protein expresses enhanced tyrosine kinase signaling activity compared to the normal ABL protein (gain of function) Feline TCRG V-N-J alignment CDR3 region Moore et al., Vet Immunol Immunopathol 106: 167-178, 2005 PCR 5’ primer 3’ V segment 3’ primer N region J segment CDR3 117 Molecular Clonality - Indications • Morphological, cytological, immuno-phenotypic properties inconclusive • Lack of architectural effacement in organized lymphoid tissue - MZL or TZL • Lympho-histiocytic proliferations in skin • Lamina proprial or intra-epithelial lymphocytosis in the small intestine Molecular clonality - Limitations • Sensitivity limited with high polyclonal background • Miss small clonal populations • Sensitivity limited- B cells - IGH V mutation • Clonality is not equivalent to malignancy • Interpret results in context • IGH and TCRG rearrangements are not markers of lineage • Cross lineage rearrangements in lymphoid and myeloid malignancies Strategies for anti-cancer immunotherapy 118 Anti-neoplastic drugs • Alkylating agents (cisplatin, cyclophosmamide etc.) – interact directly with cellular DNA, form bonds with nuclic acids and proteins • Antimetabolites (methotrexate, fluorouracil, gemcitabine etc.) – resemble cellular metabolites (folic acid, purine, pyrimidine) – interfere with DNA precursors & cellular metabolism • Antitumor antibiotics (bleomycin, doxorubicin etc.) – derived from soil fungus, some anti-infective activity – interfere with DNA activity • Mitotic inhibitors (vinblastin, paclitaxel, docetaxel etc.) – derived from plant extracts – interfere with formation of mitotic spindle, arresting mitosis • Endocrine agents (tamoxifen, prednisolone, goserelin etc. ) – aromatase inhibitors, oestrogen antagonist, corticosteroids, LHRH agonist • Molecularly targeted agents (retinoids, imatinib etc.) – gene expression, monoclonal antibody, tyrosine kinase inhibitor Sites of Action of Cytotoxic Agents PURINE SYNTHESIS •6-MERCAPTOPURINE •6-THIOGUANINE PYRIMIDINE SYNTHESIS RIBONUCLEOTIDES •METHOTREXATE •5-FLUOROURACIL •HYDROXYUREA •PEMETREXED DEOXYRIBONUCLEOTIDES ALKYLATING AGENTS AKYLATING LIKE (INTERCALATING) ANTIBIOTICS DNA •CYTARABINE •GEMCITABINE ETOPOSIDE RNA TOPOISOMER PROTEINS L-ASPARAGINASE VINCA ALKALOIDS ENZYMES MICROTUBULES TAXOIDS Acknowledgement 119 ? 120 121 122 123 124 125 126 127 128 129 130 131