Physiology 364 Cancer PDF

CANCER Lecture 1 Cancer What is cancer? Definitions: Neoplasm: new growth o Malignant neoplasm: ▪ uncontrolled growth (division beyond normal limits) ▪ invasion (intrusion on and destructi...

CANCER Lecture 1 Cancer What is cancer? Definitions: Neoplasm: new growth o Malignant neoplasm: ▪ uncontrolled growth (division beyond normal limits) ▪ invasion (intrusion on and destruction of adjacent tissues; go into other tissues) ▪ metastasis (spread to other locations in the body via blood or lymph) o Benign neoplasm: ▪ self-limited: although they proliferate, they are usually contained and surrounded by a connective tissue sheet ▪ do not invade ▪ do not metastasize Oncology: Branch of medicine concerned with the study, diagnosis, treatment and prevention of cancer Primary tumours usually spread through the blood vessels and lymph vessels to distant organs Intravasation= When primary tumour cells enter the blood vessels Extravasation= when these tumour cells exit the blood vessels 1  CANCER Aetiology For most patients the cause of illness (disease) is unknown, however, several factors have been identified as being associated with the development of the malignancy: o Cigarette tobacco o Alcohol o Diet o Exposure to ultraviolet light o Infectious agents o Drugs ▪ Oestrogens – breast, vaginal and endometrial carcinomas Causative factors associated with the development of cancer Smoking→ mouth, pharynx, oesophagus, lung, bladder lip Ultraviolet light→ skin, lip Alcohol→ mouth, pharynx, larynx, oesophagus, colorectal Drugs→ bladder, bone marrow Asbestos→ lung, mesothelium Oestrogens→ breast, endometrium, vagina Vinyl chloride→liver (angiosarcoma) Polycyclic hydrocarbons→ skin, lung Aromatic amines→ bladder Aflatoxin→ liver Biological agents associated with the development of cancer: A. Hepatitis B virus: liver B. Schistosoma japonicum (paracite): gut, bladder C. Helicobacter pylori (gram-negative bacterium): stomach D. Human papilloma virus (HPV ) – Gardasil vaccine: cervical cancer Parasite enters the body and begin to produce eggs, it uses the hosts' Gram-negative bacteria immune system (granulomas) for transportation of eggs into the gut Infection preventable by vaccination Hepatitis B virus DNA viruses 2  CANCER Epidemiology Study of the incidence and distribution of the disease Projected at 17 million deaths in 2030 Accounts for 13% of all deaths worldwide Cancer is responsible for 1 in 8 deaths worldwide Cancer causes more deaths than AIDS, tuberculosis, and malaria combined o Geographical distribution o Age distribution Oncology-related terms Tumour: solid neoplasm (neoplasms such as leukemia do not form tumours) Pre-malignancy, pre-cancer or non-invasive tumour (this is a tumour which is still confined to the tissue that it originates from) o Eg: If it’s an epithelial tumour, it will still be confined to the epithelial tissue o A neoplasm that is not invasive but has the potential to progress to cancer (become invasive) if left untreated 3  CANCER These lesions: atypia, dysplasia and carcinoma in situ (stage 0, non-invasive) o in order of increasing potential for cancer There are different classification systems – example cervical cancer, in the original classification, it was known as CIN1, CIN2, and CIN3, where CIN stands for Cervical Intraepithelial Neoplasia CIN1 is only mild dysplasia o Dysplasia= immature disordered cells (aka atypical cells) If there’s only a few atypical cells or dysplastic cells then it is known as mild dysplasia and this is equal to CIN1, if it’s moderate, CIN2 and if its severe, CIN3 If all the epithelial cells are abnormal or dysplastic or atypical, then it is known as carcinoma in situ Carcinoma in situ is also known as stage 0 o This means that it is non-invasive so it hasn’t spread to any other tissue Most recent classification for cervical intraepithelial neoplasia= LSIL and HSIL o LSIL= Low-grade squamous Intra-epithelial lesions, and HSIL= High-grade squamous Intra-epithelial lesions LSIL is equal to CIN1 and HSIL is equal to CIN 2 and 3 Focus on images on bottom left: Normal cells become cancerous via different stages Left: normal epithelial cells, then when they start increasing in number (hyperplasia) o just an increase in number of cells, is not cancer but when the cells become abnormal, this is known as dysplasia and as soon as the cells spread to different tissue, this is known as invasive cancer, which is then then classified as stage 1 to stage 4 Squamous epithelial on the top part of the images; lower pictures resemble ductal carcinoma The first one is just a normal duct which is lined by normal, simple cuboidal epithelium and then hyperplasia, intraductal hyperplasia is just an increase in the amount of cells, which is not cancer, but if there is atypical cells then the cells become cancerous Intraductal carcinoma in situ= still confined to the duct so it hasn’t spread yet and as soon as it starts spreading out of the epithelial tissue, as with invasive ductal cancer Now you can see the cancer cells have spread from the epithelial tissue to the connective tissue below= invasive ductal carcinoma or ductal cancer 4  CANCER Atypical columnar epithelial cells Screening: a test done in healthy people to detect tumours before they become apparent o A mammogram is a screening test Diagnosis: the confirmation of the cancerous nature of a lump o This usually requires a biopsy or removal of the tumour by surgery, followed by examination by a pathologist Surgical excision: the removal of the tumour by a surgeon Recurrence: a new tumour that appear at the site of the original tumour after surgery Surgical margins Evaluation by a pathologist of the edges of the tissue removed by the surgeon to determine if the tumour was completely removed (negative margins) or if tumour was left behind (positive margins) Grade A number (usually on a scale of 3) established by a pathologist to describe the degree of resemblance of the tumour to the surrounding benign tissue Grading systems: Bethesda Gleeson, etc 5  CANCER Grading of cervical intraepithelial neoplasia Stage A number (usually on a scale of 4) established by the oncologist to describe the degree of invasion of the body by the tumour Metastasis: new tumours that appear far from the original tumour Transformation: low-grade tumour can transform to a high-grade tumour over time Chemotherapy: treatment with drugs Radiation therapy: treatment with radiation Prognosis: the probability of cure after the therapy o expressed as a probability of survival five years after diagnosis or it can be expressed as the number of years when 50% of the patients are still alive 6  CANCER Modes of chemotherapy Primary chemotherapy: Used as the only anti-cancer treatment in highly sensitive tumour types Concurrent chemotherapy: Given simultaneous to radiation to increase the sensitivity of cancer cells to radiation Adjuvant chemotherapy: Given after surgical removal of tumour to “mop-up” microscopic residual disease in the knowledge that widespread microscopic dissemination occurred Neoadjuvant chemotherapy: Given before surgical removal of tumour to shrink the tumour to increase the chance of successful resection Classification Carcinoma: Malignant tumours derived from epithelial cells o Represents the most common cancers, including the common forms of breast, prostate, lung, cervical and colon cancer Sarcoma: A cancer that arises from transformed cells of mesenchymal origin o Malignant tumors made of cancerous bone, cartilage, fat, muscle, vascular, or hematopoietic tissues are, by definition, considered sarcomas Lymphoma and leukemia: malignancies derived from hematopoietic (blood-forming) cells Germ cell tumour: Tumours derived from totipotent cells o In adults most often found in testicles and ovaria; in fetuses, babies and young children most often found in the body midline Blastoma (blastic tumour): A tumour (usually malignant) which resembles immature or embryonic tissue o Many of these tumours are most common in children Human development begins when a sperm fertilizes an egg and creates a single totipotent cell o In the first hours after fertilization, the cell divides into identical totipotent cells Roughly four days after fertilization and after cycles of cell division, these totipotent cells begin to specialize Totipotent cells have total potential o They specialize into pluripotent cells that can give rise to most of the tissues needed for fetal development Pluripotent cells undergo further specialization into multipotent cells that are committed to give rise to cells that have a particular function o Eg: multipotent blood stem cells give rise to the red cells, white cells and platelets in the blood Malignant tumours: usually named using –carcinoma, -sarcoma or –blastoma as a suffix, with the Latin or Greek word for the organ of origin as the root. Eg. Cancer of: o liver – hepatocarcinoma o cancer of fat cells – liposarcoma Malignant tumours are classified as carcinomas: they originate from epithelial tissue, sarcomas from mesenchymal tissue and blastomas are from more immature or embryonic tissue o These terms form the suffix of the word, then the Latin or Creek word for the organ of origin as the root of the word ▪ Eg: cancer of the liver: It will be of epithelial origin so that’s why it’s a carcinoma and then liver is hepato= hepatocarcinoma ▪ Cancers of fat cells: Fat cells are of mesenchymal origin, that’s why it will be a sarcoma, and fat in Latin or Greek will by lipo= liposarcoma 7  CANCER Epithelia o Carcinoma – cancer of epithelial origin o Adenocarcinoma – cancer of glandular epithelium o Angiosarcoma – cancer of endothelial cells (inner surface of heart and all blood vessels) o Mesothelioma – cancers of mesothelium lining the ventral cavities of the body Connective tissue o Fibromas – benign tumours of fibroblast origin o Lipomas – benign tumours of adipose tissue o Liposarcomas – cancer of adipose tissue o Leukemias – cancers of blood-forming tissues o Lymphomas – cancers of lymphoid tissues o Chondromas – benign tumours in cartilage o Chondrosarcomas – cancer of cartilage o Osteomas – benign tumours of bone o Osteosarcomas – cancer of bone Muscle tissue o Myomas – benign muscle tumours o Myosarcomas – cancer of skeletal muscle tissue o Cardiac sarcomas – cancer of cardiac muscle tissue o Leiomyomas – benign tumours of smooth muscle o Leiomyosarcomas – cancer of smooth muscle tissue Neural tissues o Gliomas – cancer of neuroglial origin o Neuromas – cancer of neuronal origin ▪ Connective tissue has mesenchymal origin: cancers of connective tissues= sarcomas ▪ Benign connective tissue tumours: if they originate in fibroblasts, this will be known as fibromas, in fat cells they’ll be known as lipomas but if its cancer of fat cells then it will be a liposarcoma ▪ Benign tumours of cartilage= chondromas, but malignant tumours of cartilage= chondrosarcomas ▪ Benign tumours of bones= osteomas and cancer of bones will be osteosarcomas Questions & Answers 1) Name 3 properties of a malignant neoplasm? ▪ uncontrolled growth (division beyond normal limits) ▪ invasion (intrusion on and destruction of adjacent tissues) ▪ metastasis (spread to other locations in the body via blood or lymph) 2) What is the aetiology:(factors which have been identified as being associated with the development of the malignancy) and the epidemiology: (the incidence and distribution) of a disease? 3) Classification: ▪ Malignant tumour which arises from cartilage - chondrosarcoma ▪ Malignant tumour from blood-forming tissue - leukemia ▪ Benign tumour of bone - osteoma ▪ Malignant tumour from embryonic tissue of the liver - hepatoblastoma 8  CANCER Lecture 2 Signs and symptoms Local symptoms: o Unusual lumps or swelling (tumour), hemorrhage (bleeding), pain and/or ulceration o Compression of surrounding tissue may cause symptoms such as jaundice (yellowing of eyes and skin) Symptoms of metastasis (spreading): o Enlarge lymph nodes, cough, hepatomegaly (enlarged liver), bone pain, fracture of affected bones and neurological symptoms Systemic symptoms: o Weight loss, poor appetite, fatigue, cachexia (wasting), excessive sweat (night sweats), anemia and paraneoplastic phenomena, i.e. conditions that are due to the cancer such as thrombosis or hormonal changes Causes Anything which replicates will probabilistically suffer from errors (mutations) Mutations will survive and might be passed on to daughter cells Body normally safeguards against cancer via: o Apoptosis o Helper molecules (DNA polymerases): eg PARP (poly-ADP- ribose-polymerase) o Senescence (vs Quiescence) Quiescence vs Senescence Quiescence occurs due to growth factor withdrawal and is a reversible process When growth factors are added again then the cell with continue with the cell cycle It is viewed as either an extended G1 phase, where the cell is neither dividing, nor preparing to divide, or a distinct quiescence stage, which we score G0, outside the cell cycle Senescence: is an irreversible process Activated by DNA damage, leads to activation of p53, and activation of tumour suppressor genes like p21 and p16 p21= an oncogene because sometimes it will also induce tumorigenesis Markers of senescence are β-gal (beta- galactosidase) which is a staining method to observe this enzyme in the cytoplasm of the cell Another marker of senescence= senescence- 9 associated heterochromatin foci o Seen in the nucleus of the cell  CANCER Cell Cycle Phases of cell cycle: The M phase is the mitotic phase: consists of the pro-, meta, ana, and telophase o G1 phase: first growth phase where there is growth of the cells and also normal metabolic processes o S phase: DNA replication takes place o G2 phase: growth phase as well as preparation phase for mitosis You must know this diagram The cell cycle is regulated by cyclins as well as cyclin-dependent kinases; The Cyclin D is activated with the highest levels in the S and G2 phase Cyclin E is upregulated just before the S phase, Cyclin A in the G2 phase and the Cyclin B just before mitosis Explain the role and mechanism of action of retinoblastoma (Rb) as a tumour suppressor protein: The Rb protein is a tumor suppressor: plays an NB role in the negative control of the cell cycle and in tumor progression It’s responsible for a major G1 checkpoint, blocking S-phase entry and cell growth Phosphorylated retinoblastoma protein is responsible for a major G1 checkpoint, so it’s blocking the S phase entry, and therefore it’s blocking cell growth When retinoblastoma is hypo-phosphorylated, it inhibits activity of the E2F family of transcription factors The E2F family of transcription factors are needed for transcription of genes that are essential for the cell to enter the cell cycle When Rb is hyper-phosphorylated then it releases E2F, so the free E2F is essential for the initiation of the G1 phase of the cell cycle 10  CANCER More causes Error correction often fails in small ways, especially in hostile environments: o Presence of carcinogens (disruptive substances) o Periodic injury o Hypoxic environments Cancer is a progressive disease= progressive errors accumulate till cells act contrary to its normal function Clonal evolution One of the reasons it’s so difficult to treat cancer is because of the clonal evolution of cancer cells o So selective pressures allow some of the mutant subclones to expand while others go extinct Some subclones mutations will occur and more subclones will be formed, the selective pressures of the environment will allow some of them to expand and others will die This is why the single founder mutation and the mutations earlier on is not the same as the clones which arises later in different environments Branching clonal architecture of clonal evolution in cancer Selective pressures allow some mutant sub-clones to expand while others go extinct Mutation: chemical carcinogens: Substances causing DNA mutations – mutagens Mutagens which causes cancer – carcinogens Tobacco smoking – 90% of lung cancers Asbestos fibers – mesothelioma Many mutagens are also carcinogens, some carcinogens are not mutagens, eg alcohol – promote cancer by stimulating the rate of cell division, faster replication leaves less time for repair of damaged DNA o Mutagen= causes mutation of the genes Mutation: ionizing radiation: o Ultraviolet radiation o Mobile phones Viral or bacterial infection: o Hepatitis B; HPV Hormonal imbalances: o Hyperestrogenic states – endometrial cancer Immune system dysfunction: o HIV – Kaposi’s sarcoma, non-Hodgkin’s lymphoma 11 Obesity and diet  CANCER It is important to understand the relationship between excessive caloric intake and the development of cancer: High caloric intake will cause adipocytes to increase in size and number, so the hypertrophic adipocytes will reduce the secretion of adiponectin, but increase the secretion of free fatty acids as well as increasing aromatase activity o This will lead to chronic systemic low-grade inflammation which will cause hyperinsulinemia and also insulin resistance ▪ This will then decrease steroid hormone-binding globulin and also insulin-like growth factor-1 secretion from the liver, which will lead to more bioavailable IGF-1 in the circulation as well as sex hormones High levels of insulin, IGF-1 and sex hormones such as estrogens are very potent agonists for cell proliferation, where IGF-1 and insulin can bind to receptor tyrosine kinase on epithelial cells which will activate the PI3-kinase pathway, which will cause increased cell proliferation and inhibits apoptosis And then in the end this will cause genome instability which will then lead to cancer Adrenal Glucocorticoids Synthesis pathways for steroid hormones Shows where aromatase fits in Aromatase is responsible for the conversion of the male steroid hormones into the female steroid hormones 12  CANCER These pathways play a major role in cancer science In many cancers these genes are mutated in these pathways Insulin, IGF-1 and IGF-2 can bind to the same receptor, receptor tyrosine kinase So different pathways can be activated through this receptor Receptor tyrosine kinase can activate via IRS-1, the PI3-kinase pathway o PI3-kinase is responsible for the conversion of PIP2 to PIP3, and then PKB/Akt is recruited to the cell membrane, where it is phosphorylated on the serine and threonine residues The active PKB/Akt will inhibit apoptosis Activation of the same receptor through the recruitment of adaptor proteins, namely Shc and Grb2 will be able to activate Ras, Raf and then the MEK and ERK pathway which will also cause an increase in cell proliferation Syndromes of cancer with a hereditary component: BRCA1 & BRCA2: Breast and ovarian cancer MEN types 1, 2a, 2b: Multiple endocrine neoplasia TP53: Li-Fraumeni syndrome- osteosarcoma, breast cancer, soft tissue sarcoma, brain tumours Turcot syndrome: characterized by multiple adenomatous colon polyps with increased risk of colorectal cancer and brain cancer o It may be associated with: ▪ Familial adenomatous polyposis (mutation of the APC gene) ▪ Hereditary nonpolyposis colorectal cancer (HNPCC / Lynch syndrome) ▪ Glioblastoma multiforme - mutation in one of the mismatch repair genes, MLH1 and PMS2 Retinoblastoma: In children: hereditary mutation in retinoblastoma gene Down syndrome: extra chromosome 21 – develop malignancies such as leukemia and testicular cancer Attributes acquired during evolution of cancer cells Enhanced proliferation and survival Ability to overcome spatial limitations by invading surrounding tissue Survive under conditions of low oxygen and nutrients Evade host tumor defenses Travel to distant organs Resist anti-cancer treatment 13  CANCER Mechanism Most cancers develop through interaction of genetic and environmental factors Genetic factors: o Hereditary predisposition ▪ Born with genes that increase the likelihood of cancer ▪ Cancer is not guaranteed, but more likely ▪ Inherited genes affect tissues’ abilities to: metabolize toxins, control mitosis and growth, repair after injury and identify and destroy abnormal cells o Oncogene activation ▪ Cancer may also result from somatic mutations which modify genes involved in cell growth, differentiation, mitosis and apoptosis ▪ Ordinary cells are converted into cancer cells ▪ Modified genes are called ONCOGENES; the normal genes are called proto-oncogenes Oncogenes/Oncoproteins o Normally code for proliferation signals, e.g. proteins of Ras/Raf/MEK/ERK pathway and PI3 Kinase pathway o Mutations in the Ras family of proto-oncogenes (H-Ras, N-Ras, K-Ras) occur in 20-30% of human tumours o A proto-oncogene is normally in the proliferation pathway, so a proto-oncogene is an oncogene in a normal pathway ▪ As soon as that proto-oncogene becomes mutated= an oncogene Tumour suppressor proteins/genes o Code for anti-proliferation signals and proteins that suppress mitosis and cell growth/proliferation o Function: arrest progression of cell cycle to carry out DNA repair, preventing mutations from being passed to daughter cells ▪ Eg p53 has two functions: nuclear – act as transcription factor; cytoplasmic – regulating cell cycle, cell division and apoptosis ▪ Eg retinoblastoma protein (regulates cell cycle) and PTEN (inhibits cell proliferation pathway) PTEN which inhibit the PI3 kinase pathway PI3 kinase is responsible for the conversion of PIP2 to PIP3, and PTEN is the phosphatase which remove the phosphate group from PIP3 to convert it back to PIP2 o Therefore, PTEN is also a tumour suppressor protein because it inhibits the proliferation pathway ONCOGENE: A gene that played a normal role in the cell as a proto-oncogene and that has been altered by mutation and now may contribute to the growth of a tumour NB: oncogenes include: ras (a signal transduction molecule) myc (a transcription factor), src (a protein tyrosine kinase) HER-2/neu, also called erbB-2 (a growth factor receptor) hTERT (an enzyme involved in DNA replication) Bcl-2 (a membrane associated protein that prevents apoptosis) 14  CANCER A growth factor will bind to a receptor (eg. tyrosine kinase), then adaptor proteins will be recruited to the receptor and this will cause the phosphorylation and activation of the Ras/Raf/MEK/ERK pathway, which will then induce cell growth and proliferation In normal cells, the proteins in this pathway will be known as proto-oncoproteins, and their genes proto-oncogenes o As soon as these genes are mutated, which often occur in cancer, then these genes and proteins are known as proto-oncoprotein, and proto- oncogenes THE PI3-K PATHWAY: MECHANISM OF PKB/Akt ACTIVATION PI3 kinase pathway and how it is activated: A growth factor will bind to a receptor tyrosine kinase o This will lead to the phosphorylation and recruitment of PI3 kinase to the receptor o PI3 kinase is made of p85 and p110 residues Phosphorylated PI3 kinase leads to the conversion of phosphatidylinositol bisphosphate to phosphatidylinositol trisphosphate (PIP2 will be converted to PIP3) PIP3 will induce the recruitment of the inactive PKB to the cell membrane Normally PKB is in the cytosol and it’s inactive, so as soon as PIP3 is formed, PKB will be recruited to the cell membrane where it will be phosphorylated There are two residues where it is phosphorylated - the kinase domain will be phosphorylated at the threonine 308 residue by a protein (PDK-1), which is situated in the cell membrane and the regulatory loop of PKB will be phosphorylated on serine 473, by mTORC2 o These residues must become phosphorylated for PKB to become active The activated PKB can then phosphorylate other proteins downstream of this This pathway is regulated very well through the action of the phosphatases o Phosphatases inhibit this pathway to discontinue cell proliferation Two very NB phosphatases SHIP and PTEN dephosphorylate the kinase to inactivate it Remember: PI3 kinase will add a phosphate group to PIP2 to convert it to PIP3 The phosphatases, PTEN and SHIP then remove the phosphate groups to convert it back to PIP2 o These phosphatases also remove different phosphate groups from different positions on PIP3 o NB: the proteins which are in the proliferation pathways such as PI3 kinase and PKB, are proto-oncoproteins, because when they are activated they will cause cell growth and cell proliferation The phosphatases are tumour suppressor proteins because normally they will stop or inhibit the proliferation pathways 15  CANCER Mechanism of PKB activation In unstimulated cells, PKB is not phosphorylated on Thr308 or Ser473 and resides mainly in the cytosol Following growth factor (GF) activation of receptor tyrosine kinases (RTK), PI3-Kinase is recruited to the receptor and phosphorylated, resulting in the conversion of PIP2 to PIP o This recruits PKB to the membrane where it is phosphorylated by PDK-1 on Thr308 and on Ser473 by mTORC2 Active PKB is then released from the membrane and translocates to the other subcellular compartments where it can phosphorylate other proteins The threonine and the serine residue must be phosphorylated on PKB so it can become fully active The active PKB can then also induce the phosphorylation and activation of different proteins In nucleus: Forkhead transcription factors (FKHR) cause transcription of death genes o This means that these genes will induce cell death, especially apoptosis o PKB causes cell proliferation, so it must inhibit cell death, so when PKB is active it phosphorylates these Forkhead kinases which reside in the nucleus and this will lead to the translocation of these FKHR out of the nucleus to the cytosol and in the cytosol it will bind to this 14-3-3 protein which makes this inactive So now the Forkhead kinases can’t induce transcription of death genes when it’s phosphorylated by PKB The active PKB can also cause the phosphorylation of Iкβ, so usually NFкβ is in a dimer with Iкβ as soon as it’s phosphorylated, NFкβ will translocate to the nucleus and then it will induce the transcription of survival genes o Therefore, in this case it will be the inhibitor of apoptosis proteins PKB can also phosphorylate CREB o CREB will then translocate into the nucleus and will also cause transcription of survival genes In mitochondria: PKB can also phosphorylate BAD o BAD is usually residing on the mitochondrion membrane o BAD is a pro-apoptotic protein which will normally cause pore formation in the mitochondria which will cause release of cytochrome c from the mitochondria, but as soon as it’s phosphorylated by PKB, then BAD will also bind to the 14-3-3 protein which makes it inactive, therefore it cannot induce apoptosis 16  CANCER Lecture 3 Question: Identify the tumour suppressor proteins as well as the proto-oncoproteins Both of these pathways indicated on this page are cell proliferation pathways When the proteins in these pathways are phosphorylated, they are activated and results in the induction of cell proliferation o The pathways= PI3 kinase pathway as well as the RAS/RAF/MEK/ERK pathway Pathways are activated by phosphorylation and that is why in normal cells, all these proteins involved in these pathways are known as proto-oncoproteins In cancer many of these genes in these pathways are mutated o When genes are mutated, the proteins are known as oncoproteins and the genes oncogenes In normal conditions these pathways must be stopped otherwise cells will continue to proliferate Kinases are activated through phosphorylation, and for these kinases to be inactivated, they must be dephosphorylated o The phosphatases are responsible for the dephosphorylation of kinases The phosphatase which dephosphorylates PI3 kinase is known as PTEN, and the other one is SHIP They will remove a phosphate group from PIP3 to form PIP2 There function is therefore to stop the proliferation pathways, therefore they are known as tumour suppressor proteins A dual specific phosphatase or MKP-1 (MAP Kinase phosphatase-1)= Another tumour suppressor protein o Responsible for the dephosphorylation of the MAP kinases It can dephosphorylate MAP kinases and the MEKs to inactivate them MKP-1 is a phosphatase and a tumour suppressor protein, because when it is active, it halts the proliferation pathway 17  CANCER NF-κB is located in cytoplasm and an inactive state and is kept in cytoplasm by Inhibitor kappa B (IkB) proteins PI3K/Akt signalling pathway leads to phosphorylation of IKKα, which phosphorylates Ik-B proteins o Ik-B proteins phosphorylated by IKKα is exposed to proteasomal degradation, resulting in nuclear translocation and transcriptional activation of NF-κB How NF-кβ is activated: NF-кβ will be phosphorylated by PKB or Akt, then it will translocate to the nucleus, where it will induce the transcription of survival genes, like the inhibitor of apoptosis proteins Phosphorylated Akt (remember that Akt must be phosphorylated on both the serine and the threonine residues to be fully active) can then cause activation of NF-кβ Normally NF-кβ is located in the cytoplasm, in an inactive state, and it forms a dimer with inhibitor of кβ proteins After phosphorylation of IKKα by PKB/Akt, phosphorylated IKKα will phosphorylate I-β causing the release of NF-кβ from the dimer - which is now free to translocate to the nucleus to cause gene transcription. Iкβ, after it has been phosphorylated by IKKα, will then be degraded by the proteasome p53 Discovered in 1979 o Cellular protein which accumulate in nuclei of cancer cells o Gene encoding p53 – oncogenic – over-expressed in human and mouse tumour cells 10 years later researchers discover that oncogenic properties of p53 actually resulted from mutations of p53 Early 1990’s data from knockout mice – potent tumour suppressor actions of p53 The structure of p53 (Guardian of the Genome) Nuclear phosphoprotein MW(molecular weight) 53 kDa, encoded by a 20-Kb gene containing 11 exons and 10 introns, located on the small arm of chromosome 17 Belongs to highly conserved gene family containing two other members: p63 and p73 Contains 393 amino acids 18  CANCER 3 functional domains: N-terminal activation domain, DNA binding domain (central core), C-terminal tetramerization domain Demonstration of how p53 can induce DNA repair, apoptosis, senescence, and also play an important role in cell cycle checkpoints How is it possible for one protein to have these diverse range of functions? o p53 can be activated through DNA damage, heat shock, hypoxia, oncogene overexpression and many different other factors. What will determine the response of p53? o p53 has many different phosphorylation sites, so skite of phosphorylation determines if p53 will induce apoptosis, senescence or DNA You must know what are the cellular stressors that will repair induce p53 activation, and also the cellular responses which can be achieved by p53 activation Under normal conditions: p53 in short-lived and expression levels are kept extremely low MDM2 (murine double minute 2) acts as an E3 ubiquitin protein ligase for p53, promotes its ubiquitination followed by degradation by 26S proteasome In response to genotoxic stress – p53 P at multiple sites, including Ser-15, Ser-20 and Ser-46 o exerts pro-apoptotic effects There are two major protein degradation pathways o Autophagy= degrades large protein as well as cell organelles o Ubiquitin-proteasome system only degrades proteins 19  CANCER Ubiquitin-proteasome system The ubiquitin-proteasome system (UPS) and the autophagic-lysosomal pathway= two major degradation systems for both native and misfolded proteins in eukaryotic cells, which do not act independently from each other Defective autophagy results in accumulation of ubiquitinated proteins, impacting the flux of the UPS, while dysfunction of the UPS can promote a compensatory induction of autophagy Protein degradation and the maintenance of protein homeostasis results in the regulation of many normal cellular processes including signal transduction, cell cycle control, transcription, inflammation and apoptosis by the UPS The regulated proteolysis of bulk and misfolded proteins is strictly controlled by the 26S proteasome complex o The 26S proteasome complex recognizes polyubiquitinated proteins, which were marked for elimination by the E1, E2 and E3 ubiquitinating enzymes Upon recognition, unfolding and transfer of the de-ubiquitinated target protein by the 19S regulatory cap into the interior of the cylindrical 20S proteasome core particle, protein degradation is facilitated by catalytic b- subunits having nucleophilic N-terminal threonine (Thr1) residues Eukaryotic 20S proteasomes harbour seven different b-subunits in their two-fold symmetrical a7b7b7a7 stacked complexes, only three b-subunits per b-ring [subunits b1 (caspase-like), b2 (trypsin-like) and b5 (chymotrypsin- like)] are proteolytically active o These three b-subunits are major targets for small molecule proteasome inhibitors The blockade or inactivation of the 26S proteasome complex-regulated degradative process, using small molecule inhibitors against one or more catalytic b-subunits, can lead to significant build-up of cytotoxic proteins Subsequently, apoptotic pathways are activated, specifically in rapidly proliferating cells 20  CANCER P53 regulation: MDM2 acts as a ubiquitin ligase for p53 to promote itdegradation p19ARF = a tumour suppressor protein and promotes the stabilization of p53 by inactivating MDM2 Hepatoblastoma and p53 p53 is infrequently mutated in HBL– no role in development of tumours p53 is accumulated abnormally in cytoplasm of HBL – nuclear exclusion represents non-mutational mechanism of p53 inactivation, also abnormally sequestered in cytoplasm of breast and colon cancers Detailed mechanisms of accumulation of cytoplasmic p53 are unclear Parc (p53-associated parkin-like cytoplasmic protein) which interacts with p53 might inhibit its nuclear translocation Mechanism for hepatoblastoma is due to Parc o Parc interacts with p53 and inhibits its nuclear translocation o Therefore, if it cannot enter the nucleus it is unable to perform its tumour suppressor functions On the left-hand side: shows p53 in the nucleus Right-hand side: shows a p53 hepatoblastoma Only in the cytoplasm, not nucleus 21  CANCER Hanahan and Weinberg (2000) summarized biological properties of cancer cells: Acquisition of self-sufficiency in growth signals: leads to unchecked growth Loss of sensitivity to anti-growth signals: leas to unchecked growth Loss of capacity for apoptosis, in order to allow growth despite genetic errors and external anti-growth signals Loss of capacity for senescence: leads to limitless replicative potential (immortality) Acquisition of sustained angiogenesis, allowing the tumour to grow beyond the limitations of the passive nutrient diffusion Acquisition of ability to invade neighbouring tissues= defining property of invasive carcinoma Acquisition of ability to metastasize to distant sites Loss of capacity to repair genetic errors – increased mutation rate Hallmarks of Cancer 22  CANCER Prevention of cancer Modify lifestyle risk factors Alcohol consumption Smoking Obesity Inactivity Use of exogenous hormones Ultraviolet radiation Diet Obesity – increased risk for breast cancer Reduced meat consumption – reduces colon cancer risk Moderate coffee consumption – reduced risk of stomach cancer Plant-based diet – reduced prostate and breast cancer risk Curcumin (tumeric); resveratrol, omega-3 fatty acids – anticancer effects High consumption of refined sugars and simple carbohydrates – increased risk of cancer Vitamins Beta-carotene – protective effect Some actions we can take to reduce some of the risk factors for cancer: Chemoprevention Daily use of tamoxifen (selective estrogen receptor modulator – SERM) reduced the risk of breast cancer in high- risk women COX-2 inhibitors, eg. rofecoxib and celecoxib reduced risk of colon polyp incidence in familial adenomatous polyposis patients Genetic testing BRCA1, BRCA2 – breast, ovarian, pancreatic cancers MLH1, MSH2, MSH6, PMS1, PMS2 – colon, uterine, stomach, urinary tract Vaccination HPV vaccine for cervical cancer – Cervarix and Gardasil o The HPV virus is associated with cervical cancer, vaccines for the prevention of cervical cancer= Cervarix and also Gardasil Screening Detect unsuspected cancer in asymptomatic population Early diagnosis – extended life Mammography, Pap-smear 23  CANCER Rofecoxib and celecoxib, the cyclooxygenase inhibitors and specifically the COX-2 inhibitors are acting within the cell o The cell membrane consists of phospholipids which have fatty acids attached to the carbon backbone Different fatty acids are attached to the positions, Sn-1, Sn-2 and Sn-3 on the phospholipid o When phospholipase A2 is activated, the fatty acids which is in the Sn-2 position of the phospholipid will be released o That fatty acid will then through the action of the cyclooxygenases, convert to cyclic endoperoxides, which consist of prostaglandins, prostacyclins and thromboxanes Rofecoxib and celecoxib (reduce the risk of colon cancer) act as cyclooxygenase inhibitors Diagnosis Investigation Blood tests X-rays CT scans Endoscopy MRI (Magnetic Resonance Imaging – magnetic fields & radio waves) Histological examination Biopsy of cancerous cells (tissue intact) are cut and stained and examined by pathologists Cytology Cervical smear/Pap smear -loose cells are scraped from cervix, stained and examined under microscope by pathologist Screening for cancer: Males: prostate, colon Females: Breast, Ovary, Cervix, Colon Screening tests: Prostate: PSA blood test and rectal exam Colon: Colonoscopy, Fecal occult blood test Breast: Mammogram Cervix: Pap/Cervical smears Ovary: Blood test and ultrasound of the pelvis 24  CANCER Treatment Surgery Removal of tumour Staging for prognosis and adjuvant therapy Palliative treatment: control symptoms such as spinal cord compression Radiation therapy Ionizing radiation to kill cancer cells and shrink tumours Externally: external beam radiotherapy (EBRT) Localised and confined to area being treated Damaging genetic material of cells – impossible to grow and divide External beam radiotherapy Chemotherapy Cytotoxic drugs which affect rapidly dividing cells Problem= affects normal cells with high replacement rate (eg. intestinal lining) o Causes nausea Leukemias and lymphomas requires high-dose chemotherapy and total body irradiation - treatment ablates bone morrow Harvesting of blood stem cells before therapy autologous stem cell transplantation o some of the bone blood stem cells are harvested before therapy and then they inject it into the patient again after therapy Hematopoietic stem cells transplanted from matched unrelated donor (MUD)= allogeneic stem cell transplantation 25  CANCER Targeted therapies Agents specific for deregulated proteins of cancer cells Small molecule targeted therapy: o Inhibitors of enzymatic domains on mutated, over-expressed and other critical proteins in cancer cells, eg tyrosine kinase inhibitors, imatinib Monoclonal antibody therapy: o Ab which binds to specific protein on surface of cancer cell, eg anti-HER2/neu antibody, trastuzumab (HERCEPTIN), used in breast cancer Targeted Therapy Immunotherapy Induce patient’s own immune system to fight tumour, eg interferons and other cytokines to induce an immune response in renal cell carcinoma and melanoma patients. Immune checkpoint inhibitors 26  CANCER Allogeneic hematopoietic stem cell transplantation (HSCT) Bone marrow transplantation from genetically non-identical donor Donor’s immune cells will attack the tumour (graft-versus-tumour-effect) Allogeneic HSCT leads to higher cure rate than autologous transplantation – although more severe side effects Hormonal therapy Used in breast (ER+, PR+, HER2+ - Herceptin) and prostate cancers, eg hormone antagonists for breast cancer or anti-androgens for prostate cancer Upon ligand binding, dimerization between receptors of Epidermal growth factor receptor (EGFR) family and Human Epidermal growth factor receptor 2 (HER2 receptor) is induced The homodimers or heterodimers thereafter stimulate a serial of signalling cascades HER family signalling and targeted therapies in breast cancer HER family receptors form homo- and hetero-dimers at the cell surface, which can be induced either dependently or independently of ligand: 27  CANCER Angiogenesis inhibitors Prevent the extensive growth of blood vessels (angiogenesis) that tumours require to survive, eg bevacizumab are in clinical use o Problems with anti-angiogenesis drugs: ▪ many factors stimulate blood vessel growth in normal and cancer cells ▪ anti-angiogenesis drugs only target one factor; other factors continue to stimulate blood vessel growth ▪ route of administration, maintenance of stability and activity and targeting tumour vasculature Clinical Staging Why staging? To determine whether it has spread, to prescribe the best /most appropriate treatment Staging is specific to specific types of cancer o Prostate cancer – Gleason system o Cervical cancer – Bethesda system Universal TNM system o T = primary tumour size (T0-T4) ▪ T0: absence of primary tumour, T4 largest dimension and greatest amount of invasion o N = lymph node involvement (N0-N3) ▪ NO: no lymph node invaded by cancer cells, N1 involvement of single lymph node less than 3 cm in diameter, N2 presence of one medium-sized node (3-6 cm) or multiple nodes smaller than 6 cm, N3 single lymph node larger than 6 cm, whether or not other nodes are involved. o M = extent of metastasis (M0 or M1) ▪ M0: no evidence of metastasis, M1 cancer cells have produced secondary tumours in other part of the body Clinical Staging and tumour grading Correlation between tumour staging and prognosis/treatment o T low stage: no lymph node involvement, good cure rate o T high stage: lymph node involvement, metastasis, poor survival rate o Early stage: surgery usually leads to cure o T3, T4, N1 or N2: radiation and/or chemotherapy useful in addition to surgery o M1: treatment can prolong life Limitless replicative potential Telomeres Simple-sequence DNA repeats at end of chromosomes Each cell division: 50-100bp of telomeric DNA are lost from ends of every chromosome Eventually lose ability to protect ends of chromosomes – death of affected cell Telomere maintenance in malignant cells 85-90% upregulation of telomerase Unlimited multiplication of descendant cells 28  CANCER Malignant Melanomas Melanomas are one of the most malignant types of cancers with the highest death rate Etiology of melanomas Genetic factors: o Mutations in tumor suppressor genes and proto-oncogenes, eg. CDKN2A (p16, p19), CDK4, RB1, PTEN and ras o CDKN2A (p16): Tumor suppressor gene located on band 9p21, mutation play a role in various cancers o Important in sporadic and hereditary melanomas Ultraviolet radiation: o Exposure to ultraviolet radiation (UVR) o UVA (wavelength 320-400 nm) and UVB (wavelength 290-320 nm): ▪ suppression of immune system of skin ▪ induction of melanocyte division ▪ free radical production ▪ damage to melanocyte DNA Sunburn: o Acute, intense and intermittent blistering sunburns on areas only receiving occasionally sun exposure Other factors: viruses, chemicals, changing mole, family history 29  CANCER Staging of the melanomas: 30  CANCER Lecture 4 Test a novel compound X for its anti-cancer properties You are a researcher in Dr Smith’s laboratory at Oxford University and are given a novel compound to test for its anti-cancer properties. What signaling pathways will most probably be affected by such a compound and what experiments would you recommend? The PI3-k pathway: mechanism of PKB/AKT activation Growth factor will bind to receptor tyrosine kinase o Causes phosphorylation of the receptor and PI3 kinase will be recruited to the receptor and phosphorylated PI3 kinase then causes the conversion of PIP2 to PIP3, through phosphorylation, a phosphate group will be added to PIP2 to form PIP3 o This will be the signal for inactive PKB in the cytoplasm to translocate to the cell membrane In the cell membrane there’s PDK-1 and also mTORC2, PDK-1 can phosphorylate the threonine residue of PKB and mTORC2 will phosphorylate the serine residue of PKB o These residues must be phosphorylated for PKB to be fully active NB: a kinase is active when it is phosphorylated, and a phosphatase is active when it’s dephosphorylated The phosphatases (PTEN and SHIP) remove the phosphate groups that the kinases have added o PTEN and SHIP remove phosphate groups on different sites Also remember, since these phosphatases act to inhibit this proliferation pathway, they are also tumour suppressor proteins 31  CANCER What the active PKB can do in the cell to prevent cell death and to induce cell survival The active PKB can phosphorylate different proteins The Forkhead kinases are usually in the nucleus of the cell and they are responsible for the transcription of death genes PKB will phosphorylate these Forkhead kinases and then they will move out of the nucleus into the cytoplasm, and there they will be bound to 14-3-3 protein o When a protein binds to 14-3-3 protein, it renders it inactive so it will stay in the cytoplasm and it will not be able to induce transcription of death genes PKB will also phosphorylate IKKα, and IKKα will then phosphorylate Iкβ In the cytoplasm Iкβ and NFкβ form a dimer, but as soon as it’s phosphorylated by IKKα, then NFкβ can translocate into the nucleus, where it can cause the transcription of survival genes PKB can also phosphorylate CREB, CREB will then translocate to the nucleus and also cause the transcription of survival genes In mitochondria: there’s a protein on the mitochondrial membrane which is known as BAD o This is a pro-apoptotic protein which form pores in the mitochondrial membrane during apoptosis But when PKB phosphorylates BAD, it will also bind to 14-3-3 protein then BAD cannot make pores in the mitochondrial membrane to induce apoptosis Necrosis vs Apoptosis Apoptosis: an energy-dependent process Initially the sarcolemma and mitochondria will be preserved, there will be chromatin condensation and then apoptotic bodies will be formed These small bodies are found close to the cell, and these apoptotic bodies will be removed by macrophages or neighbouring cells No inflammation is involved in this process Necrosis: the opposite of apoptosis, swelling takes place There will be no more ATP left in the celldisruption of the sarcolemma and the mitochondria, chromatin clumping and blebbing takes place and then there will be inflammation Morphological criteria Necrosis ▪ Swelling ▪ Depletion of ATP ▪ Disruption of sarcolemma & mitochondria ▪ Chromatin clumping & blebbing ▪ Inflammation Apoptosis ▪ Energy-dependent ▪ Preservation of sarcolemma & mitochondria ▪ Chromatin condensation ▪ Removal by macrophages & neighboring cells 32  CANCER The molecular pathways through which apoptosis is induced Two molecular pathways through which apoptosis can be induced in the cell: Death receptor pathway: a ligand, usually a death ligand like TNF-α, or Fas ligand binds to its receptor and this will induce apoptosis in the cell Mitochondrial pathway: activated if the stress factor is inside the cell, while the death receptor pathway will be activated if it’s outside the cell Both of these two pathways end on the caspases which are the proteases of the apoptotic pathway Apoptosis Receptor mediated pathways: a ligand bind to the receptor will then induce cleavage of caspase 8 o The caspases indicated in purple blocks are the initiator caspases o The initiator caspases must first be activated and then they will activate the effector caspases The effector caspases are caspases 3,6 and 7; caspase 12, 8 and 9 are initiator caspases The mitochondrial pathway is activated when there’s some damage inside the cell such as DNA damage, then the JNK/p53 pathway will be activated o This induces pore formation in the mitochondria, and then cytochrome c will be released and the apoptosis pathway will continue After the cleavage of the effector caspases, these caspases can then cause cleavage of PARP o PARP (Poly-ADP-ribose polymerase) PARP is a repair enzyme which will repair the DNA damage o During apoptosis, if the effector caspases, cleave PARP then PARP will not be able to perform its function, and then the cell will undergo apoptosis which will lead to DNA fragmentation, cell shrinkage, membrane blebbing, and the formation of apoptotic bodies, which will then be removed by macrophages or neighbouring cells 33  CANCER When conducting an experiment, we must have a hypothesis, aims and also experimental procedure: Hypothesis o Compound X will induce apoptosis in the cancer cells and alter the PI-3kinase signaling pathway Aims o Evaluate the chemotherapeutic / antiproliferative potential of compound X on a colon cancer cell line, and to test its effect on normal cells o To investigate the role of the PI3-K pathway in this phenomenon Experimental Procedures ✓ Cells were incubated for 24h with Compound X ✓ Harvest proteins for SDS PAGE & Western blot analysis: o PKB/Akt Ser473; Thr308; PI3-K, PTEN, BAD, CREB, caspase-3 PARP ✓ MTT assay for cell viability ✓ Fluorescence microscopy: Hoechst staining for apoptosis, PI for necrosis When experiments are done on cancer cells, we must also use a normal cell line to see if the compound damages the normal cells as well We use CaCo cells, which are colon cancer cells and the NCM 460 cells which is the normal colon epithelial cell line and which also serves as our control experimental group Fluorescence microscopy will be used and stain for apoptosis markers as well as necrosis markers An MTT viability assay will be done and then we must determine which proteins are activated in the PI3-kinase pathway, e.g. PKB, PI3-kinase, PTEN, Bad, CREB, caspase-3 and PARP SDS PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) Determine the proteins in our experiment: Mobility of substance in gel: influenced by charge and size SDS disrupts the secondary, tertiary & quaternary structure of the protein to produce linear polypeptide 34  CANCER Results The effect of compound X on cell viability in cancer cells and normal colon cells The first experiment is to conduct a MTT cell viability assay to determine the effects of this compound on normal cells as well as on cancer cells We then use different concentrations to see which concentrations we must further include in our experimental setup On the left-hand side you will see the bar graphs which indicate the normal epithelial cells and the effect of the compound on these cells, even the high concentrations did not have any effect on the normal epithelial cells On the right-hand side you will see that all three of the concentrations significantly decreased cell viability 120 p