Physiology 364_ Cancer.pdf

Physiology 364: Cancer Lecture 1 What is cancer? Cancer is a disease characterized by the uncontrolled growth and spread of abnormal cells. These cells can form a mass called a neoplasm or tumor. Neoplasms can be classified as either benign or malignant. Definitions: Neoplasm: A new and abnor...

Physiology 364: Cancer Lecture 1 What is cancer? Cancer is a disease characterized by the uncontrolled growth and spread of abnormal cells. These cells can form a mass called a neoplasm or tumor. Neoplasms can be classified as either benign or malignant. Definitions: Neoplasm: A new and abnormal growth of tissue in some part of the body. Malignant neoplasm (cancer): ○ Uncontrolled growth: Cells divide beyond normal limits, continuing to grow and multiply without the usual regulatory mechanisms. ○ Invasion: Cancer cells intrude on and destroy adjacent tissues. ○ Metastasis: Cancer cells spread to other locations in the body via the blood or lymphatic system. Benign neoplasm: ○ Self-limited: Growth is limited and does not continue indefinitely. ○ Do not invade: Benign tumors do not intrude on or destroy adjacent tissues. ○ Do not metastasize: Benign tumors do not spread to other parts of the body. Oncology: This is the branch of medicine concerned with the study, diagnosis, treatment, and prevention of cancer. (What is metastis) Tissue invasion and metastasis, is the most heterogeneous and poorly understood. It is, however, critically important because metastasis accounts for most cancer fatalities. Distant metastases are tumour cells that have broken away from the primary tumour,  have traveled to other parts of the body, and have begun to grow at the new location. In most cases there is not a continuous trail of tumour cells between the primary site and the distant site. Tissue invasion is extension from the primary organ beyond adjacent tissue into the next organ; for example, from the lung through the pleura into bone or nerve. (Intravasation and Extravasation) NPs might help breast cancer cells enter and leave the bloodstream by weakening the blood vessel barrier. In an experiment, researchers tested this in female mice. They implanted a tumor in the mammary fat and injected NPs into the mice's tail veins. The NPs might interact with blood vessel linings in the primary tumor, making the vessel walls leakier by disrupting connections between endothelial cells. This would allow cancer cells to leave the tumor (intravasation) and enter the bloodstream, increasing the number of circulating tumor cells (CTCs). Cancer cells leaving the bloodstream can form new tumors (extravasation). General Causes Several factors have been identified as being associated with the development of malignancies: - Cigarette tobacco - Alcohol - Diet - Exposure to ultraviolet light - Infectious agents - Drugs - Oestrogens (which are linked to breast, vaginal, and endometrial carcinomas) Subtances/chemicals that can cause cancer Biological agents that can cause cancer: These biological agents can contribute to cancer development through various mechanisms such as chronic inflammation, direct cellular damage, and persistent infection leading to cellular mutations. A. Hepatitis B Virus (HBV) - Associated Cancer: Liver cancer (hepatocellular carcinoma) - Explanation: Hepatitis B virus is a DNA virus that infects the liver. The virus causes persistent inflammation and cell damage, leading to the accumulation of mutations in liver cells that can result in cancer. Vaccination against HBV can significantly reduce the risk of liver cancer. B. Schistosoma japonicum (Parasite) - Associated Cancers: Gut cancer (including colorectal cancer) and bladder cancer - Explanation: Schistosoma japonicum is a parasitic worm that causes schistosomiasis. Chronic infection with this parasite leads to inflammation and scarring of the affected tissues. In the gut, it can cause chronic inflammation and fibrosis, which increases the risk of developing colorectal cancer. In the bladder, it can cause schistosomiasis-related inflammation and damage, which increases the risk of bladder cancer. C. Helicobacter pylori (Gram-Negative Bacterium) - Associated Cancer: Stomach cancer (gastric cancer) - Explanation: Helicobacter pylori (H. pylori) is a bacterium that infects the stomach lining, causing chronic gastritis and peptic ulcers. Chronic infection with H. pylori can lead to inflammation and damage to the stomach lining. Over time, this persistent inflammation can lead to the development of gastric cancer. Eradication of H. pylori infection with antibiotics has been shown to reduce the risk of developing stomach cancer. D. Human Papillomavirus (HPV) - Associated Cancer: Cervical cancer - Explanation: Human papillomavirus (HPV) is a group of viruses with many types, some of which are classified as high-risk for causing cancer. Persistent infection with high-risk HPV types, especially HPV-16 and HPV-18, is a major cause of cervical cancer. The virus causes changes in the cells of the cervix, leading to dysplasia and potentially cancer. The Gardasil vaccine is designed to protect against the most common high-risk HPV types and thus significantly reduces the risk of cervical cancer. Epidemiology Definition: Epidemiology is the study of how often diseases occur in different groups of people and why. For cancer, this involves examining the incidence (new cases) and distribution (how cases are spread across different populations) of cancer. Projected Statistics for 2030: 17 million deaths: It's estimated that cancer will cause 17 million deaths worldwide by 2030. 13% of all deaths: Cancer is projected to account for 13% of all deaths globally. 1 in 8 deaths: Cancer is responsible for 1 out of every 8 deaths worldwide. Cancer causes more deaths than AIDS, tuberculosis, and malaria combined. Geographical Distribution: Cancer incidence and mortality rates can vary significantly between different regions and countries. For example, some cancers may be more prevalent in low-  and middle-income countries due to differences in healthcare access, environmental factors, and infectious agents. Age Distribution: Cancer Risk by Age: The risk of developing cancer generally increases with age. Most cancers are diagnosed in older adults, as cancer often takes years to develop and manifest. However, children can develop cancer as well. Oncology-related terms (1): Tumour: A solid neoplaasm or solid mass of abnormal cells. However, Leukemia, which is a type of cancer that affects blood cells does not form solid tumours. Pre-malignancy, pre-cancer, or non-invasive tumour: A neoplasm that is not yet cancerous but could become invasive if not treated. Types include: - Atypia: Minor abnormalities in cell appearance. - Dysplasia: More significant abnormalities, indicating a higher risk of cancer. - Carcinoma in situ (stage 0): A localized and non-invasive cancer that has not spread. LSIL (Low-Grade Squamous Intraepithelial Lesions): A mild form of dysplasia in the cervical cells that is often associated with HPV infection and has a low risk of progressing to cancer. CIN (Cervical Intraepithelial Neoplasia): A classification of cervical cell changes with CIN 1 being low-grade and CIN 2-3 being high-grade, which have increasing potential to progress to cervical cancer if untreated. Oncology-related terms (2) Screening: a test done in healthy people to detect tumours before they become apparent. A mammogram is a screening test. Diagnosis: the confirmation of the cancerous nature of a lump. 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 Surgical margins refer to the edges of the tissue removed during surgery. A pathologist examines these margins to ensure that no cancer cells remain. Negative margins: No cancer cells are found at the edges, indicating the tumour has been completely removed. Positive margins: Cancer cells are present at the edges, suggesting that some tumour may have been left behind. Grade Grade: A score (often on a scale of 1 to 3) given by a pathologist to indicate how much a tumour resembles the surrounding normal tissue. Lower grades generally mean the tumour looks more like normal tissue, while higher grades indicate it looks less like normal tissue and may be more aggressive. Grading systems: - Bethesda: Often used for classifying cervical lesions. - Gleeson: Commonly used for grading prostate 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. Tumor Stage (WHO) ○ Stage 0-microscopic disease only ○ Stage 1-tumor confined to the dermis ○ Stage II-tumor does not infiltrate subcutaneous tissues, lymph node metastasis ○ Stage III-large, infiltrating tumor (or multiple tumors) ○ Stage IV-distant metastasis Consideration is being given to reducing stage of multiple dermal tumors Oncology-related terms (3) 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 Immunotherapy: Cancer Prac  Prognosis: the probability of cure after the therapy. It is usually 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. Modes of chemotherapy…the basics Primary chemotherapy ○ Used as the sole 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 (1) Carcinoma: Malignant tumours derived from epithelial cells. 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. Thus, 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. 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. Many of these tumours are most common in children. Human development begins when a sperm fertilizes an egg and creates a single totipotent cell. In the first hours after fertilization, this cell divides into identical totipotent cells.  Approximately four days after fertilization and after several cycles of cell division, these totipotent cells begin to specialize. Totipotent cells have total potential. They specialize into pluripotent cells that can give rise to most, but not all, of the tissues necessary for fetal development. Pluripotent cells undergo further specialization into multipotent cells that are committed to give rise to cells that have a particular function. For example, multipotent blood stem cells give rise to the red cells, white cells and platelets in the blood. Classification (2) Malignant tumours are named with specific suffixes and a root indicating their origin: carcinoma: Used for cancers originating in epithelial tissues. Example: Hepatocarcinoma (liver cancer). sarcoma: Used for cancers originating in connective tissues like bone, muscle, or fat. Example: Liposarcoma (cancer of fat cells). blastoma: Used for cancers originating from immature or embryonic cells. Example: Neuroblastoma (cancer of nerve cells). Classification (3) Epithelia: - Carcinoma: Cancer originating from epithelial cells. - Adenocarcinoma: Cancer of glandular epithelial tissue. - Angiosarcoma: Cancer of endothelial cells (lining blood vessels and the heart). - Mesothelioma: Cancer of the mesothelium (lining of body cavities). Connective Tissue: - Fibromas: Benign tumours from fibroblasts (connective tissue cells). - Lipomas: Benign tumours of adipose (fat) tissue. - Liposarcomas: Cancer of adipose tissue. - Leukemias: Cancers of blood-forming tissues (like bone marrow). - Lymphomas: Cancers of lymphoid tissues (like lymph nodes). - Chondromas: Benign tumours in cartilage. - Chondrosarcomas: Cancer of cartilage. - Osteomas: Benign tumours of bone. - Osteosarcomas: Cancer of bone. Classification (4) Muscle Tissue: - Myomas: Benign tumours of muscle tissue. - Myosarcomas: Cancer of skeletal muscle tissue. - Cardiac Sarcomas: Cancer of cardiac (heart) muscle tissue. - Leiomyomas: Benign tumours of smooth muscle (e.g., in the uterus). - Leiomyosarcomas: Cancer of smooth muscle tissue. Neural Tissues: - Gliomas: Cancers originating from neuroglial cells (supportive cells in the nervous system). - Neuromas: Cancers originating from neuronal cells (nerve cells). Lecture 2 Signs and symptoms 1. Local symptoms: a. Unusual lumps or swelling (tumour), hemorrhage (bleeding), pain and/or ulceration. b. Compression of surrounding tissue may cause symptoms such as jaundice (yellowing of eyes and skin). 2. Symptoms of metastasis (spreading) a. Enlarge lymph nodes, cough, hepatomegaly (enlarged liver), bone pain, fracture of affected bones and neurological symptoms. 3. Systemic symptoms: a. 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 (1) 1. Replication Errors: Anything that replicates can suffer from errors, known as mutations. 2. Mutation Survival: Mutations can survive and potentially be passed on to daughter cells. 3. Body's Safeguards Against Cancer: a. Apoptosis: Programmed cell death to eliminate damaged cells.  b. Helper Molecules: DNA polymerases correct replication errors. c. Senescence vs. Quiescence: Senescence permanently stops cell division in damaged cells, while quiescence is a temporary state of inactivity. (Understanding Senescence vs. Quiescence) Quiescence (G0 Phase) Reversible State: Quiescence is a temporary, reversible state where cells exit the cell cycle. Occurs in G1 Phase: Happens before the restriction (R) point in the G1 phase. Mechanism: Mediated by p27-dependent CDK inactivation and cyclin D1 downregulation. Gene Expression: Certain genes in quiescent cells can block the onset of senescence (e.g., HES1). p53 Role: p53 is a tumor suppressor protein that plays a critical role in preventing cancer formation. It is often called the "guardian of the genome" because of its role in preserving stability by preventing genome mutation. Function: p53 is activated in response to various cellular stresses such as DNA damage, oxidative stress, and oncogene activation. Upon activation, p53 can lead to cell cycle arrest, DNA repair, apoptosis (programmed cell death), or senescence. p21 Role: p21 (also known as CDKN1A) is a cyclin-dependent kinase inhibitor. Function: p21 is a downstream target of p53. When p53 is activated, it can induce the expression of p21. p21 binds to and inhibits the activity of CDKs, particularly CDK2 and CDK4/6, leading to cell cycle arrest at the G1 phase. This allows the cell to repair DNA damage or, if the damage is too severe, to undergo senescence or apoptosis. p16 Role: p16 (also known as CDKN2A) is another cyclin-dependent kinase inhibitor. Function: p16 inhibits CDK4 and CDK6, preventing these kinases from phosphorylating the retinoblastoma protein (Rb). This inhibition leads to cell cycle arrest in the G1 phase. p16 is often involved in the regulation of cellular senescence and is activated in response to various stress signals, including those not directly involving p53. CDKs (Cyclin-Dependent Kinases) Role: CDKs are a family of protein kinases involved in regulating the cell cycle. Function: CDKs, when complexed with their regulatory cyclin partners, phosphorylate target proteins that drive the cell cycle progression from one phase to another (e.g., G1 to S phase, G2 to M phase). CDKs are essential for the orderly progression of the cell cycle, but their activity must be tightly regulated to prevent uncontrolled cell proliferation. Cellular Senescence Definition: Cellular senescence is a state of stable cell cycle arrest where cells no longer proliferate but remain metabolically active. It is a mechanism to prevent the propagation of damaged or stressed cells, acting as a barrier to cancer development. Mechanism: Senescence can be induced by various factors, including DNA damage, telomere shortening, oxidative stress, and activation of oncogenes. p53 and p16 pathways are crucial in mediating the onset of senescence. Activation of p53 leads to the expression of p21, which inhibits CDKs and causes cell cycle arrest. Similarly, activation of p16 also inhibits CDKs, particularly CDK4 and CDK6, leading to cell cycle arrest and senescence. Recap 1. Stress Response and p53 Activation: Cellular stress activates p53. 2. p53 Induces p21: Activated p53 induces the expression of p21. 3. p21 Inhibits CDKs: p21 inhibits CDK2 and CDK4/6, leading to cell cycle arrest in the G1 phase. 4. p16 Inhibits CDKs: Independent of p53, p16 can inhibit CDK4 and CDK6, also leading to cell cycle arrest in the G1 phase. 5. Senescence Induction: Both p53-p21 and p16 pathways contribute to the induction of cellular senescence by halting cell cycle progression, thereby preventing damaged or stressed cells from proliferating. Key Differences - Reversibility: Quiescence is reversible, while senescence is irreversible. - Mechanisms and Markers: Different molecular pathways and cell markers distinguish the two states. (Cell Cycle) There are 3 check points in the cell cycle; G1, G2 and M signals registered at the checkpoint report the status of the various cellular conditions checkpoints integrate a variety of internal (intracellular) and external (extracellular) information for many cells, the G1 checkpoint, is the most important a go ahead signal usually indicates that the cell will complete the cycle and divide in the abscence of this signal, the cell may exit the cell cycle, switching to the nondividing state called G0 phase (Understanding Cyclins and Cdks in the Cell Cycle) Cyclin Concentrations and Checkpoints Cyclin Levels Change: The concentrations of cyclin proteins fluctuate throughout the cell cycle. Checkpoints Correlation: Cyclin accumulation is directly related to the three major cell cycle checkpoints.  Cyclin Degradation: Cyclin levels drop sharply after each checkpoint as they are broken down by cytoplasmic enzymes. Role of Cyclins and Cdks Cyclins and Cdks: Cyclins regulate the cell cycle only when bound to Cyclin-dependent kinases (Cdks). Activation: The Cdk/cyclin complex must be phosphorylated at specific sites to be fully active. Phosphorylation changes the shape of proteins, activating them. Function: Activated Cdks phosphorylate other proteins, helping the cell advance to the next phase. Stable Cdk Levels: The levels of Cdk proteins remain relatively constant, while cyclin levels fluctuate. Specific Binding: Different cyclins bind to Cdks at specific points in the cell cycle, regulating different checkpoints. Explain the role and mechanism of action of retinoblastoma (Rb) as a tumour suppressor protein The Rb protein is a tumor suppressor, which plays a pivotal role in the negative control of the cell cycle and in tumor progression. Rb protein (pRb) is responsible for a major G1 checkpoint, blocking S-phase entry and cell growth. (Effects of Cyclins on the Cell Cycle) 1. Cyclin-CDK Complexes:  - Cyclin D binds with CDK4/6. - Cyclin E binds with CDK2. - These complexes phosphorylate the Rb protein, activating it. 2. Role of Rb Protein: - Rb is a tumor suppressor that controls cell cycle progression. - When phosphorylated, Rb releases E2F, which activates genes needed for the G1/S transition. 3. Regulation Pathways: - Cyclin D1 can be regulated by the RAS-MEK-ERK pathway (transcription level) and by mTOR via S6K and 4EBP1 (translation level). - mTOR inhibitors can decrease Cyclin D1 action. - CDK4/6 inhibitors can reduce the effects of Cyclin D1, especially if CDK4/6 is amplified or CDKN2A/B is lost (CDKN2A/B normally inhibits CDK4/6). 4. Cancer Implications: - Loss of Rb function is common in cancer. - Patients with Rb loss or mutations might be resistant to mTOR or CDK4/6 inhibitors. Causes (2) 1. Hostile Environments and Error Correction Failure: In hostile environments, such as those with carcinogens, hypoxia, or frequent injuries, the body's mechanisms for correcting cellular errors can fail. These environmental stressors increase the likelihood of errors during cell division and DNA repair, contributing to the development of cancer. 2. Clonal Evolution: Cancer progresses as cells with genetic mutations that provide a growth advantage proliferate more than normal cells. Over time, these mutated cells accumulate additional errors, further deviating from normal function and leading to the aggressive and uncontrolled growth characteristic of cancer. (Natural Selection) The classical model of clonal evolution, proposed by Nowell, describes how cancer progresses through the sequential acquisition of mutations, leading to successive dominance of different sub-clones (selective sweeps). Disease progression from adenoma to carcinoma to metastasis supports this view. During this process, individual cells and their sub-clones compete for space and resources. Analyzing mutations in single cells over time is the best way to study this clonal architecture, though there are few examples so far. These studies show complex patterns of mutation segregation, consistent with Nowell's model. Overall, tissue and single-cell analyses confirm that cancer evolution is complex and branching, similar to Darwin's evolutionary tree Cell division: Normal cell vs cancerous cell Causes (3) Mutation:chemical carcinogens: ○ Substances causing DNA mutations – mutagens ○ Mutagens which causes cancer – carcinogens ○ Tabacco 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. Mutation: ionizing radiation: ○ Ultraviolet radiation ○ Mobile phones Viral or bacterial infection: ○ Hepatitis B; HPV Hormonal imbalances: ○ Hyperestrogenic states – endometrial cancer Immune system dysfunction: ○ HIV – Kaposi’s sarcoma, non-Hodgkin’s lymphoma Obesity, diet (Effects of Excessive Calorie Intake and Adiposity on Hormones and Cell Proliferation) Effects of excessive calorie intake and adiposity on hormones and growth-factor production and cell proliferation Excessive calorie intake and a sedentary lifestyle promote hypertrophy of adipose tissue, reduce adiponectin production, and increase circulating free fatty acids (FFA) and inflammation, leading to insulin resistance and compensatory hyperinsulinemia. Increased serum insulin concentration causes a reduction in hepatic synthesis of insulin-like growth factor binding protein 1 (IGFBP1) and steroid hormone binding globulin (SHBG), that leads to increased bioavailability of insulin growth factor 1 (IGF-1) and sex hormones. Adipose tissue is also a major source of extra-glandular estrogens. Chronically elevated circulating levels of insulin, IGF-1, sex hormones and inflammatory cytokines promote cellular proliferation, genomic instability, and inhibit apoptosis in many cell types. IGF-1R Signaling Pathway: Activation: IGF-1, IGF-2, or insulin binds to the IGF-1R α-subunit. Autophosphorylation: This binding causes the β-subunit to autophosphorylate. Docking Site Formation: The phosphorylated β-subunit becomes a docking site for insulin receptor substrates (IRS-1 to 4). PI3K Pathway: IRS-1 activates PI3K, which then activates Akt. Inhibition by PTEN: PTEN inhibits PI3K. Apoptosis Inhibition: Activated Akt inhibits apoptosis by inactivating BAD. Protein Synthesis Regulation: Akt phosphorylates TSC1/2, relieving their inhibition of mTOR. ○ mTOR Activation: mTOR activates S6K and 4E-BP-1, leading to protein synthesis. Energy Depletion Response: When energy is low, LKB1 and AMP levels increase, activating AMPK. AMPK's Role: AMPK inhibits mTOR directly or indirectly by activating TSC1/2. Hypoxia Response: Hypoxia induces REDD1, which interacts with TSC1/2, though the details are unclear. TSC1/2 might inhibit HIF-α independently of REDD1. MAPK Pathway: IGF-1R activation also triggers the MAPK pathway. Activation Sequence: IGF-1R activates adaptor proteins Shc and Grb2, leading to the activation of Ras, Raf, MEK1/2, and ERK1/2. Outcome: This pathway results in cell proliferation Causes (4) 1. Hereditary Breast and Ovarian Cancer: - BRCA1 & BRCA2 Genes: Mutaions increase the risk of breast and ovarian cancer, often found in families with a strong history of these cancers. 2. Multiple Endocrine Neoplasia (MEN): - MEN Type 1: Tumors in parathyroid glands, pituitary gland, and pancreas. - MEN Type 2A: Medullary thyroid carcinoma, pheochromocytoma, and parathyroid hyperplasia. - MEN Type 2B: Similar to Type 2A with additional mucosal neuromas. 3. Li-Fraumeni Syndrome: - TP53 Gene Mutation: Increases risk of multiple cancers, including osteosarcoma, breast cancer, soft tissue sarcoma, and brain tumors, often at a young age. 4. Turcot Syndrome: - Multiple adenomatous colon polyps and increased risk of colorectal and brain cancer. - Familial Adenomatous Polyposis (FAP): Mutation in APC gene, leading to many colon polyps and high colorectal cancer risk. - Hereditary Nonpolyposis Colorectal Cancer (HNPCC/Lynch Syndrome): Mutations in mismatch repair genes like MLH1 and PMS2, associated with glioblastoma multiforme and other cancers. 5. Retinoblastoma: - Rb Gene Mutation: Leads to retinoblastoma, a rare eye cancer in children, often affecting one or both eyes at a very young age. 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 Mechanism Genetic and Environmental Interactions: Most cancers result from a combination of genetic predispositions and environmental factors. Genetic Factors: Hereditary Predisposition:  ○ Germline Mutations: Inherited genes increase cancer risk but do not guarantee it. ○ Impact: These genes affect how tissues metabolize toxins, control growth and mitosis, repair injuries, and identify and destroy abnormal cells. Oncogene Activation: ○ Somatic Mutations: These mutations change genes involved in cell growth, differentiation, mitosis, and apoptosis, turning ordinary cells into cancer cells. ○ Oncogenes vs. Proto-oncogenes: Oncogenes are mutated forms of proto-oncogenes, which are normal genes that regulate cell growth. ○ Oncoproteins: These proteins, such as those in the Ras/Raf/MEK/ERK and PI3K pathways, signal cell proliferation. Mutations in the Ras family occur in 20-30% of human tumors. Tumor Suppressor Proteins/Genes: ○ Function: These genes produce signals and proteins that prevent excessive cell growth and proliferation. ○ Role: They arrest the cell cycle to repair DNA, preventing mutations from being passed on. ○ Examples: p53: Acts as a transcription factor in the nucleus and regulates the cell cycle, cell division, and apoptosis in the cytoplasm. Retinoblastoma Protein (Rb): Regulates the cell cycle. PTEN: Inhibits cell proliferation pathways. Abnormal Cell Growth Experimental data from Ductal Carcinoma In Situ (DCIS) show that 2-3% of cases develop metastasis after "curative" treatment despite no residual primary tumor. This inquiry is based on that finding. Tumor suppressor genes regulate cell division. When mutated, these genes fail to control cell growth, potentially leading to cancer. For example, a mutation in the BRCA1 gene increases the risk of breast cancer. Tumor suppressor genes act like brakes in a car, while oncogenes act like the accelerator. Overexpression of oncogenes, such as HER2-neu in breast and stomach cancer or the MYC gene in lymphomas, promotes cancer formation. Oncogenes are mutated proto-oncogenes that contribute to tumor growth. Key oncogenes include: - ras (signal transduction molecule) - myc (transcription factor) - src (protein tyrosine kinase) - HER2/neu (growth factor receptor) - hTERT (DNA replication enzyme) - Bcl-2 (protein preventing apoptosis) Mechanism of PKB activation brief overview. (More detail to come) 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 PIP3.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. (Fig. shows the MAPK (Mitogen-Activated Protein Kinase) cascades in mammals) These pathways involve a series of proteins and kinases working in a three-tiered sequence: 1. Ras (a small GTPase) activates 2. Raf (MAP3K, or MAP kinase kinase kinase), which then activates 3. MEK (MAP2K, or MAP kinase kinase), leading to 4. ERK (MAPK, or extracellular signal-regulated kinase). This stepwise activation also occurs in other MAPK cascades. The diagram includes GPCRs (G-protein coupled receptors) involved in initiating the cascade. The PI3K pathway (and here is the more detail) The PI3K pathway is a critical signaling pathway in cells that regulates a variety of cellular processes, including growth, survival, and metabolism. The activation of PKB/Akt through this pathway involves several key components and steps. Growth Factor (GF) Binding to Receptor Tyrosine Kinase (RTK): ○ The pathway is typically initiated when a growth factor (GF) binds to its specific receptor on the cell surface, which is often a receptor tyrosine kinase (RTK). ○ Binding of the GF causes dimerization and autophosphorylation of the RTK, creating docking sites for downstream signaling molecules. 2. Recruitment and Activation of PI3 Kinase: ○ The PI3 kinase (PI3K) is recruited to the phosphorylated RTK via its regulatory subunit (p85), which binds to the phosphotyrosine residues on the RTK. ○ The p85 subunit is associated with the catalytic subunit (p110) of PI3K. Once recruited to the membrane, PI3K is activated. 3. Production of PIP3: ○ Activated PI3K phosphorylates the membrane phospholipid PIP2 (phosphatidylinositol 4,5-bisphosphate) to produce PIP3 (phosphatidylinositol 3,4,5-trisphosphate). ○ PIP3 serves as a second messenger that recruits proteins with pleckstrin homology (PH) domains to the plasma membrane. 4. Role of SHIP and PTEN:  ○ SHIP (SH2 domain-containing inositol-5'-phosphatase) and PTEN (phosphatase and tensin homolog) are negative regulators of the pathway. ○ SHIP dephosphorylates PIP3 at the 5' position to produce PI(3,4)P2. ○ PTEN dephosphorylates PIP3 at the 3' position to produce PIP2, thereby reducing PIP3 levels and attenuating the signaling pathway. 5. Recruitment and Activation of PDK1 and PKB/Akt: ○ PDK1 (3-phosphoinositide-dependent kinase-1) and PKB/Akt both contain PH domains that bind to PIP3, localizing them to the membrane. ○ PDK1 phosphorylates PKB/Akt on the Thr308 residue, which is critical for its activation. 6. Phosphorylation of Ser473 by mTORC2: ○ For full activation of PKB/Akt, a second phosphorylation event is required at the Ser473 residue. ○ This phosphorylation is mediated by the mTORC2 complex (mammalian target of rapamycin complex 2). 7. Activated PKB/Akt: ○ Once fully phosphorylated, PKB/Akt is fully activated and can dissociate from the membrane to phosphorylate a variety of downstream targets involved in cell survival, growth, proliferation, and metabolism. PKB (Protein Kinase B), also known as Akt. Its activation (phosphrylation) and subsequent signaling impacts multiple downstream targets, including Bad, CREB, and FKHR. Bad is a pro-apoptotic member of the Bcl-2 family. The activation of PKB/Akt inactivates Bad leading to cell survival (Bad needs to be dephosphorylated to be activated). CREB is a transcription factor that regulates the expression of genes involved in cell survival, proliferation, and differentiation. When phosphorylated by the activation f PKB/Akt, the cell survives. FKHR is a transcription factor that regulates the expression of genes involved in apoptosis, cell cycle arrest, and metabolism. When PKB/Akt is activated it inactivates FKHR which prevents apoptosis (FKHR needs to be dephosphorylated to be activated). Lecture 3 Activation Mechanisms of PI3K/Akt/NF-κB Signaling Pathway: Let’s recap what we know: Once fully phosphorylated, PKB/Akt is fully activated and can dissociate from the membrane to phosphorylate a variety of downstream targets involved in cell survival, growth, proliferation, and metabolism. Activated PKB/Akt influences several downstream components, including NF-κB, GSK-3β, mTOR, MDM2, BAD, and p27, affecting cell cycle, survival, apoptosis, metabolism, protein translation, and motility. (All this can be seen in the last picture of lecture 2) NF-κB Regulation: NF-κB is normally inactive and retained in the cytoplasm by Inhibitor kappa B (IkB) proteins. Activation of the PI3K/Akt pathway leads to phosphorylation of IKKα, which in turn phosphorylates IkB proteins. This  process results in the degradation of IkB, allowing NF-κB to move to the nucleus and activate transcription. In other words it promotes cell survival by inhibiting apoptosis through anti-apoptotic gene expression. NF-κB has been linked to lung cancer invasion and is further regulated by PTEN inactivation through the PI3K/Akt/NF-κB pathway. p53: Discovery: Identified in 1979. Function: A protein that accumulates in the nuclei of cancer cells, influencing cell behavior. Gene Encoding: The gene for p53 can be oncogenic (cancer-promoting) when over-expressed in tumors. Mutation: Initially thought to be cancer-causing, but later research showed that p53's oncogenic properties are due to mutations in the p53 gene. Tumor Suppression: Data from knockout mice in the early 1990s revealed that p53 acts as a potent tumor suppressor, helping prevent cancer development. Normal p53 = goodbye cancer Mutated p53 = hello cancer Structure of p53: Type: Nuclear phosphoprotein. Molecular Weight: 53 kDa. Gene: Encoded by a 20-kilobase gene with 11 exons and 10 introns, located on the small arm of chromosome 17. Family: Part of a conserved gene family, including p63 and p73. Amino Acids: 393. Functional Domains: ○ N-terminal Activation Domain: Initiates transcriptional activity. ○ DNA Binding Domain: Central core that binds to DNA. ○ C-terminal Tetramerization Domain: Allows p53 to form a tetramer, crucial for its function. P53 as a tumour suppressor Function: Acts as a transcription factor to regulate genes involved in the cell cycle. It maintains cell integrity by responding to DNA damage and controlling cell processes. Mechanism: p53 can suppress tumours by: ○ Activating DNA repair proteins. ○ Halting the cell cycle at the G1/S checkpoint to prevent damaged DNA from being replicated. ○ Inducing apoptosis (cell suicide) if the damage is too severe to repair. Under Normal Conditions: p53: Generally present at low levels and has a short lifespan. MDM2: A protein that acts as an E3 ubiquitin ligase for p53, tagging it for degradation by the 26S proteasome.  In Response to Genotoxic Stress: ○ p53 Activation: It is phosphorylated at multiple sites (e.g., Ser-15, Ser-20, Ser-46), which enhances its ability to induce apoptosis and manage cellular stress. Ubiquitin-Proteasome System (UPS): Role: The UPS and the autophagic-lysosomal pathway are the two main systems that degrade proteins in eukaryotic cells. They work together, not independently. If autophagy fails, ubiquitinated proteins can accumulate, affecting UPS function. Conversely, UPS issues can trigger increased autophagy. Function: The UPS maintains protein balance by regulating various cellular processes, including signal transduction, cell cycle control, transcription, inflammation, and apoptosis. 26S Proteasome Complex: This complex controls the breakdown of proteins. It recognizes proteins tagged with ubiquitin (a marker for destruction) and processes them for degradation. Process: ○ Recognition: Polyubiquitinated proteins are identified by the 26S proteasome. ○ Unfolding and Transfer: The protein is unfolded and transferred to the 20S core particle by the 19S regulatory cap. ○ Degradation: Inside the 20S core, proteins are degraded by catalytic β-subunits (b1, b2, b5), which have specific enzymatic activities. Proteasome Inhibitors: Small molecules that inhibit these β-subunits can block protein degradation, causing a build-up of harmful proteins and triggering apoptosis, especially in fast-growing cells. (Mdm2) The p53 protein, a key tumor suppressor, mainly acts as a transcription factor. This means it can bind to specific parts of DNA and activate genes that have p53 response elements in their promoters. However, p53’s activity is blocked by a protein called Mdm2. Mdm2 can attach to p53, preventing it from activating gene transcription, and also targets p53 for degradation by the proteasome. This makes p53 both inactive and unstable when bound to Mdm2. A protein called p19ARF can disrupt the binding between p53 and Mdm2. By binding to Mdm2, ARF prevents Mdm2 from interacting with p53, thus allowing p53 to become active again. When cells encounter DNA damage, they activate several checkpoints. If the damage is repairable, the cell will stop its cycle to prevent passing on mutations. If the damage is too severe, the cell will undergo programmed cell death, or apoptosis, to avoid further problems. Hepatoblastoma (HBL) Hepatoblastoma (HBL) is a type of liver cancer that rarely involves mutations in the p53 gene, which is a common mutation in many other cancers. Instead of being mutated, p53 is often found abnormally accumulating in the cytoplasm of HBL cells. This accumulation happens because p53 is excluded from the nucleus, which is a non-mutational way of inactivating it. Similar abnormal cytoplasmic sequestration of p53 is seen in some breast and colon cancers as well. The exact mechanisms behind this cytoplasmic accumulation are not fully understood, but it is thought that a protein called Parc (p53-associated parkin-like cytoplasmic protein) might be involved in preventing p53 from entering the nucleus. Hanahan and Weinberg (2000) summarized biological properties of cancer cells: Acquisition of self-sufficiency in growth signals, leading to unchecked growth Loss of sensitivity to anti-growth signals, also leading 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, leading 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, the defining property of invasive carcinoma Acquisition of ability to metastasize to distant sites Loss of capacity to repair genetic errors – increased mutation rate Schematic view of available strategies for therapeutic targeting of the various 'hallmarks of cancer' with drugs that interfere with each of the acquired capabilities necessary for tumour growth and progression. Prevention of cancer (1) Preventing cancer involves modifying lifestyle risk factors: - Alcohol Consumption: Limit intake to reduce cancer risk. - Smoking: Avoid smoking to lower the risk of various cancers. - Obesity: Maintain a healthy weight to reduce cancer risk, such as breast cancer. - Inactivity: Engage in regular physical activity to lower cancer risk. - Exogenous Hormones: Be cautious with hormone use, as it may impact cancer risk. - Ultraviolet Radiation: Protect skin from excessive sun exposure to reduce skin cancer risk. Dietary Factors: - Obesity: Linked to higher breast cancer risk. - Reduced Meat Consumption: Lowers risk of colon cancer. - Moderate Coffee Consumption: May reduce stomach cancer risk. - Plant-Based Diet: Associated with lower risks of prostate and breast cancer. - Curcumin (Turmeric), Resveratrol, Omega-3 Fatty Acids: May have anticancer effects. - High Refined Sugars and Carbohydrates: Increased risk of cancer. Vitamins: -Beta-carotene may have a protective effect. Prevention of cancer (2) Chemoprevention: Using medications to reduce cancer risk. - Tamoxifen: A selective estrogen receptor modulator (SERM) that lowers breast cancer risk in high-risk women. - COX-2 Inhibitors: Drugs like rofecoxib and celecoxib that reduce colon polyp incidence in patients with familial adenomatous polyposis (a condition linked to APC gene mutations). Genetic Testing: Identifying genetic predispositions to cancer. - BRCA1 and BRCA2: Associated with increased risks of breast, ovarian, and pancreatic cancers. - MLH1, MSH2, MSH6, PMS1, PMS2: DNA mismatch repair genes linked to higher risks of colon, uterine, stomach, and urinary tract cancers. Vaccination: Preventing cancer through vaccines. - HPV Vaccines (Cervarix and Gardasil): Protect against human papillomavirus, which can lead to cervical cancer. Screening: Early detection of cancer in asymptomatic individuals. - Mammography: For breast cancer. - Pap Smear: For cervical cancer. Early detection can improve survival rates. Arachidonic acid Diagnosis Investigation Techniques: ○ Blood Tests: Analyze blood samples for markers or abnormalities. ○ X-rays: Use radiation to view internal structures. ○ CT Scans: Provide detailed cross-sectional images using X-rays. ○ Endoscopy: Insert a tube with a camera to view internal organs. ○ MRI: Uses magnetic fields and radio waves to create detailed images of soft tissues. Histological Examination: Biopsy samples are cut, stained, and examined by pathologists for cancerous cells. Cytology: Examines cells from samples like Pap smears under a microscope. Screening for Cancer: Males: Prostate: PSA blood test and rectal exam. Colon: Colonoscopy, fecal occult blood test. Females: Breast: Mammogram. Ovary: Blood test (CA-125) and pelvic ultrasound. Cervix: Pap/Cervical smears. Colon: Colonoscopy, fecal occult blood test. Treatment (1) - Surgery: - Tumor Removal: Physically removes the cancerous tumor. - Staging: Determines the cancer stage for prognosis and further treatment. - Palliative Treatment: Manages symptoms, such as spinal cord compression. - Radiation Therapy: - Ionizing Radiation: Kills cancer cells and shrinks tumors. - External Beam Radiotherapy (EBRT): Targets specific areas, damaging the genetic material of cancer cells to prevent them from growing and dividing. - Chemotherapy: - Cytotoxic Drugs: Target rapidly dividing cells, including cancer cells. - High-Dose Chemotherapy: Used for leukemias and lymphomas, often combined with total body irradiation. - Stem Cell Transplantation: - Autologous: Harvesting and returning the patient’s own blood stem cells.  - Allogeneic: Transplanting hematopoietic stem cells from a matched unrelated donor (MUD). - Targeted Therapies: - Specific Agents: Target deregulated proteins in cancer cells. - Small Molecule Targeted Therapy: Inhibitors like tyrosine kinase inhibitors (TKIs), e.g., imatinib (Gleevec). - Monoclonal Antibody Therapy: Antibodies bind to specific proteins on cancer cell surfaces, e.g., trastuzumab (Herceptin) for HER2-positive breast cancer. Targeted Therapy Treatment (2) Immunotherapy for cancer involves stimulating the patient's immune system to fight the tumor: 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 Allogeneic hematopoietic stem cell transplantation (HSCT) - Bone Marrow Transplantation: From a genetically non-identical donor. - Graft-Versus-Tumor Effect: Donor's immune cells attack the tumor. - Higher Cure Rate: Compared to autologous transplantation, but with more severe side effects. Treatment (3) Hormonal Therapy for cancer involves: - Usage: - Breast Cancer: For ER+, PR+, and HER2+ cancers, using hormone antagonists or Herceptin (for HER2+). - Prostate Cancer: Using anti-androgens. The problem: 1. Signal Input: - When a ligand (a signaling molecule) binds to receptors in the EGFR family and the HER2 receptor, they pair up (dimerize). 2. Signal Processing: - The paired receptors (dimers) activate several signaling pathways inside the cell. The most important ones are: - PI3K Pathway: Promotes cell growth and survival. - MAPK Pathway: Encourages cell division and prevents cell death. 3. Signal Output: - These signaling pathways lead to cellular processes like tumor growth and resistance to cell death. Treatment: In breast cancer, HER family receptors can form homo- and hetero-dimers on the cell surface, which can occur either in the presence or absence of a ligand. This dimerization activates the receptors through transphosphorylation of tyrosine residues, initiating downstream signaling pathways that promote cancer cell growth and survival. Targeted therapies like trastuzumab and pertuzumab are monoclonal antibodies that bind to the HER2 receptor's extracellular domain, preventing dimerization and subsequent signaling. Trastuzumab emtansine (T-DM1) combines trastuzumab with a derivative of maytansine; upon binding to HER2, the complex is internalized and degraded in lysosomes, releasing DM1 to disrupt microtubule polymerization by binding to tubulin. Additionally, tyrosine kinase inhibitors (TKIs) such as neratinib, lapatinib, and AZD8931 (sapitinib) inhibit the activity of EGFR and HER2, blocking critical signaling pathways inside the cell. Key molecules involved in these pathways include ERK, FOXO, GSK3, MDM2, MEK, mTOR, and PKC, which are crucial for cell proliferation and survival. Treatment: Angiogenesis inhibitors - Function: These drugs prevent the growth of new blood vessels (angiogenesis) that tumors need to survive. An example in clinical use is bevacizumab. - Challenges: - Multiple Growth Factors: Many factors stimulate blood vessel growth, both in normal and cancer cells. Angiogenesis inhibitors typically target only one factor, allowing other factors to continue promoting blood vessel growth. - Drug Administration: Issues include how the drug is given, maintaining its stability and activity, and effectively targeting the blood vessels of the tumor. Clinical Staging Purpose: Determines if the cancer has spread to guide the best treatment options. Specificity: Staging systems vary by cancer type. ○ Prostate Cancer: Uses the Gleason system. ○ Cervical Cancer: Uses the Bethesda system. Universal TNM System: ○ T (Primary Tumor Size): T0: No primary tumor. T1-T4: Increasing tumor size and invasion. ○ N (Lymph Node Involvement): N0: No lymph node involvement. N0: No lymph node involvement. N1: Single node < 3 cm. N2: One medium node (3-6 cm) or multiple nodes < 6 cm. N3: Single node > 6 cm, with or without other nodes involved. ○ M (Metastasis): M0: No metastasis. M1: Metastasis present, secondary tumors in other parts of the body. Clinical Staging and tumour grading Clinical Staging and tumour grading are key concepts in cancer diagnosis and treatment:  Clinical Staging: This describes the extent of cancer based on its size (T), lymph node involvement (N), and presence of metastasis (M). It's usually denoted as stages I through IV. Tumour Grading: This indicates how abnormal cancer cells look compared to normal cells, which helps predict how quickly the cancer might grow and spread. Correlation between tumour staging and prognosis/treatment: T low stage (early stage): Tumour is small and localized, often without lymph node involvement. Typically, surgery alone can cure the cancer, leading to a high cure rate. T high stage (advanced stage): Tumour is larger or has spread to lymph nodes (N1, N2) or beyond (metastasis, M1). Treatment usually includes a combination of surgery, radiation, and/or chemotherapy. ○ Early stage: Surgery alone can often lead to a cure. ○ T3, T4, N1, N2: Requires additional treatment such as radiation and/or chemotherapy alongside surgery for effective management. ○ M1 (metastatic cancer): Treatment focuses on prolonging life and managing symptoms, rather than curative intent. Limitless replicative potential- Telomeres - Telomeres: These are repetitive DNA sequences at the ends of chromosomes that protect them from deterioration or fusion with neighboring chromosomes. Cell Division: Each time a cell divides, a small portion of the telomeric DNA (about 50-100 base pairs) is lost. Over time, this results in the telomeres becoming too short to effectively protect the chromosome ends. Cell Death: When telomeres become too short, the chromosome ends are no longer protected, leading to cell senescence (aging) or death. Malignant Cells: Cancer cells often (85% to 95 %) maintain their telomeres by upregulating the enzyme telomerase. Telomerase: This enzyme extends telomeres, allowing cancer cells to bypass normal cellular aging processes and continue to divide indefinitely. Unlimited Multiplication: Due to the continuous activity of telomerase, cancer cells can multiply without the typical limitations imposed by telomere shortening. Malignant Melanomas Etiology of melanomas Genetic Factors: Mutations: Alterations in genes such as CDKN2A (p16), CDK4, RB1, PTEN, and ras can drive melanoma development. CDKN2A (p16): This gene normally inhibits the cell cycle by preventing CDK4/6 from binding to cyclin D1, which keeps the Rb tumor suppressor protein in a hypo/unphosphorylated state and E2F inactive. Mutations in CDKN2A can disrupt this regulation, promoting uncontrolled cell growth. Ultraviolet Radiation (UVR): UVA (320-400 nm) and UVB (290-320 nm): UVR damages skin by suppressing the immune response, inducing melanocyte (pigment cell) division, and causing free radical production that damages DNA in melanocytes. Sunburn: Acute, intense, and intermittent sunburns, especially on areas that rarely get sun exposure, increase melanoma risk. Other Factors: Viruses, chemicals, changes in moles, and family history can also contribute to melanoma risk. Lecture 4: Necrosis vs Apoptosis (morphological criteria) Necrosis: Swelling: Cells and organelles swell up. Depletion of ATP: Energy stores are exhausted. Disruption of sarcolemma & mitochondria: Cell membrane and mitochondria are damaged. Chromatin clumping & blebbing: DNA becomes clumped, and the cell membrane forms blebs. Inflammation: Cellular debris triggers an inflammatory response. Apoptosis: Energy-dependent: Requires ATP to proceed. Preservation of sarcolemma & mitochondria: Cell membrane and mitochondria remain intact. Chromatin condensation: DNA condenses into dense patches. Removal by macrophages & neighboring cells: Dead cells are engulfed and removed cleanly without causing inflammation. (The molecular pathways through which apoptosis is induced) The Bcl-2 family of proteins controls cell death (apoptosis) by managing how substances pass through the mitochondria and by regulating the release of a protein called cytochrome C. Anti-apoptotic proteins: Bcl-2 and Bcl-xL, found in the outer mitochondrial membrane, prevent the release of cytochrome C, thus stopping apoptosis. Pro-apoptotic proteins: Bad, Bid, Bax, and Bim move to the mitochondria when they receive death signals, promoting the release of cytochrome C and triggering apoptosis. Here's how some specific proteins work: Bad: It forms a death-promoting complex with Bcl-xL in the mitochondria, but survival signals can stop this by causing Bad to stay in the cytosol. Bid: After being cut by caspase 8 due to Fas signaling, its active part (tBid) moves to the mitochondria. Bax and Bim: They move to the mitochondria in response to stress signals, such as the absence of survival factors. p53: Activated by DNA damage, it increases Bax production. Once cytochrome C is released, it binds to Apaf1, which then activates caspase 9, leading to cell death. Mitochondria play a key role in programmed cell death, responding to both external (extrinsic) and internal (intrinsic) signals: Extrinsic pathway: Involves Fas receptor activation, leading to Bid cleavage and the activation of Bax and Bak. Intrinsic pathway: Triggered by stresses like growth factor withdrawal, which activates JNK and deactivates Bcl-2. The balance between Bax and Bcl-2 levels determines how likely a cell is to undergo apoptosis. While the exact details of how mitochondrial permeability and cytochrome C release are regulated are still debated, Bcl-xL, Bcl-2, and Bax are believed to influence a channel (VDAC) that controls cytochrome C release. Research Plan: Testing Compound X for Anti-Cancer Properties Research Context: You are a researcher at Stellenbosch University tasked with testing a novel compound, Compound X, for its anti-cancer properties. You will investigate which signaling pathways are likely affected by this compound and recommend appropriate experiments. Hypothesis: Compound X will induce apoptosis in cancer cells and alter the PI3K signaling pathway. Aims:  1. Evaluate the chemotherapeutic/antiproliferative potential of Compound X on a colon cancer cell line. 2. Test the effect of Compound X on normal cells. 3. Investigate the role of the PI3K pathway in the action of Compound X. Experimental Procedures: 1. Treatment: ○ Treat CaCo2 cells (colon cancer cells) and NCM 460 cells (normal cells) with 10-100 µg/ml Compound X. ○ Incubate cells for 24 hours with Compound X. 2. Apoptosis and Necrosis Detection: ○ Fluorescence Microscopy: Use Hoechst staining to detect apoptosis. Use Propidium Iodide (PI) staining to detect necrosis. 3. Cell Viability Assay: ○ Perform MTT assay to measure cell viability after treatment with Compound X. 4. Protein Analysis: ○ Harvest proteins from treated cells. ○ Perform SDS PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) to separate proteins based on size. ○ Conduct Western blot analysis to detect the following proteins: PKB/Akt (Ser473, Thr308) PI3K PTEN BAD CREB Caspase-3 PARP Notes on SDS PAGE: SDS PAGE separates proteins based on their size, with smaller proteins moving faster through the gel. SDS disrupts protein structures, producing linear polypeptides, allowing for size-based separation. By conducting these experiments, you will assess the anti-cancer potential of Compound X and its impact on the PI3K signaling pathway, providing insights into its mechanism of action. RESULTS Compound X results in more Apoptosis as yu can see in the above growth and therefore prevents cancer. PI3-Kinase is less phophorylated and therefore less activated which activates pro apoptopic factors like Bad. PTEN (Phosphatase and Tensin Homolog): Role: PTEN acts as a tumor suppressor by dephosphorylating PIP3, thus inhibiting the PI3K/Akt pathway. Low Levels: Decreased PTEN activity (e.g., reduced phosphorylation of PTEN) results in increased PI3K/Akt pathway activity, which usually suppresses apoptosis. However, if PTEN is low and the PI3K/Akt pathway is less active, it may lead to increased apoptosis through reduced inhibition of pro-apoptotic factors. PKB (Akt): Role in Apoptosis: PKB (Akt) is a key regulator of cell survival and proliferation. Its activation usually promotes cell survival by inhibiting pro-apoptotic signals. Low Phosphorylation Impact: When PKB is not phosphorylated (or is phosphorylated at low levels), its activity is reduced. This means that it cannot effectively suppress pro-apoptotic factors, leading to an increase in apoptosis. CREB (cAMP Response Element-Binding Protein): Role in Apoptosis: CREB is a transcription factor involved in promoting cell survival and growth. It helps regulate the expression of genes that prevent apoptosis. Low Phosphorylation Impact: Reduced phosphorylation of CREB impairs its ability to activate survival genes. Consequently, this leads to a decrease in anti-apoptotic signals and an increase in apoptosis. FKHR (FoxO1): Role in Apoptosis: FKHR (FoxO1) is a transcription factor that, when activated (i.e., dephosphorylated), promotes apoptosis by upregulating pro-apoptotic genes and downregulating survival genes. Low Phosphorylation Impact: Low levels of FKHR phosphorylation lead to its activation and nuclear translocation, where it can initiate the expression of pro-apoptotic genes and enhance apoptosis. BAD: Role in Apoptosis: BAD is a pro-apoptotic protein that promotes apoptosis by inhibiting anti-apoptotic Bcl-2 family proteins. Low Phosphorylation Impact: When BAD is not phosphorylated (or is phosphorylated at low levels), it remains active and promotes apoptosis by enhancing the pro-apoptotic signaling pathway. More cleavage means more apoptosis and this prevents Cancer. Conclusions Compound X decreases PKB, CREB, FKHR and BAD phosphorylation Compound X ­increases apoptosis Decreases Cell viability in cancer cells Do not harm normal cells Lecture 5 The Solid Tumor Microenvironment: Causes, Consequences, and Therapy Hypoxia and Angiogenesis Hypoxia: ○ Cause: As tumors grow, they often outpace their blood supply, leading to areas with low oxygen (hypoxia). ○ Consequence: Hypoxic conditions can make tumor cells more aggressive and resistant to treatment. Angiogenesis: ○ Cause: To survive and continue growing, tumors promote the formation of new blood vessels (angiogenesis) to improve their oxygen and nutrient supply. ○ Consequence: New blood vessels support further tumor growth and can facilitate metastasis (spread of cancer). Therapy:  Anti-angiogenic drugs: These therapies aim to block the formation of new blood vessels, starving the tumor of nutrients and oxygen. Hypoxia-targeted treatments: These strategies seek to exploit the unique conditions of hypoxic tumor regions to selectively kill cancer cells. By understanding and targeting the specific conditions of the tumor microenvironment, such as hypoxia and angiogenesis, therapies can become more effective. Cancer & the solid tumour Solid Tumors: These are complex, organ-like structures that are not uniform in composition. Composition: ○ Cancer Cells: Less than half of the tumor’s volume is made up of cancer cells. ○ Blood Vessels: Only 1% to 10% of the tumor consists of blood vessels. ○ Stromal Cells: The tumor also includes non-cancerous cells such as fibroblasts and inflammatory cells, which can vary within different parts of the same tumor. Role of Normal Cells: Normal cells, including those in the stroma, contribute to the tumor’s growth and development by supporting the cancer cells and participating in the tumor’s progression. Genotype vs. Phenotype / Tumor Microenvironments Genetic Mutations: ○ Identical Mutations: Patients with the same genetic mutations can exhibit different clinical features or outcomes. ○ Different Mutations: Conversely, different mutations may lead to similar solid tumor microenvironments in various patients. Uncertainty: It remains unclear whether the observed differences in clinical features arise from environmental factors or specific genetic mutations. Implication: The tumor microenvironment should be a crucial consideration in the treatment and study of solid tumors, as it significantly influences tumor behavior and patient outcomes. The solid tumour microenvironment Tumor Stroma: This includes the altered extracellular matrix and fibroblasts, which produce growth factors, chemokines, and adhesion molecules that support tumor growth. Tumor Growth: As tumors grow, their cells can become physically separated from the surrounding tissue's blood supply, leading to limited access to nutrients and oxygen. This can create areas of hypoxia and affect tumor progression The solid tumour microenvironment: Consequences The distance of tumour cells from blood vessels has two major consequences. 1 ) Decreased delivery of valuable nutrients to tumour cells – this leads to a hypoxic gradient, radiating away from the blood vessels. 2 ) Decreased removal of waste products away from cells – this leads to increased acidity in areas that are removed from blood vessels. The tumor microenvironment's dynamics are closely linked to the partial pressure of oxygen (pO₂) and the distance from the nearest blood vessel. (A) A cartoon typically illustrates how oxygen levels and pH vary within the tumor based on proximity to blood vessels. (B) Graphs often show how pO₂ and pH gradients change with distance from the blood vessel. As you move farther from the vessel, oxygen levels decrease and acidity often increases. This creates a challenging environment for tumor cells, influencing their behavior and treatment response. The solid tumour microenvironment: pH High Glycolytic Rate: Cancer cells often break down glucose at a high rate, producing lactic acid and CO₂. Acidic Environment: Inefficient removal of these by-products leads to the accumulation of H+ ions, causing the tissue pH to become more acidic, particularly in large or poorly perfused tumors. Effects of Low pH: Acidic conditions can inhibit cell proliferation, DNA synthesis, glycolysis, and metastasis The solid tumour microenvironment: Hypoxia Hypoxia in solid tumors occurs when the demand for oxygen exceeds what the tumor's blood vessels can supply. Tumors often have limited blood vessels, leading to two types of hypoxia: 1. Chronic or Diffusion-Limited Hypoxia: Tumor cells located far from blood vessels (100-400 μm away) receive less oxygen, creating a constant low-oxygen environment. This can occur in tumors as small as 0.5 mm in diameter. 2. Transient Hypoxia: Temporary reductions in blood flow can cause brief periods of low oxygen. These conditions impact tumor growth and response to treatment. Hypoxic inducible factor (HIF) 1. 5% O2 (40 mmHg) activates HIF 2. VHL – von Hippel-Lindau protein (tumor-suppressor protein) 3. VEGF – Vascular endothelial growth factor Under normal oxygen levels (normoxia), HIF-1α is hydroxylated by prolyl-4-hydroxylase, leading to its binding with the VHL protein and subsequent degradation by the ubiquitin proteasome system. In hypoxic conditions, the hydroxylase enzyme is inactive, causing HIF-1α to remain stable and avoid degradation. This stable HIF-1α moves to the nucleus, pairs with HIF-1β to form the HIF-1 complex, which binds to hypoxia response elements (HREs) in genes. This interaction alters gene expression, notably increasing the production of vascular endothelial growth factor (VEGF), which promotes the growth of new blood vessels (neoangiogenesis). Vascular endothelial growth factor (VEGF) Angiopoietin-2 (Ang-2) and VEGF – angiogenic factors induced by HYPOXIA Figure 5 shows how HIF-1α, VEGF, and growth factors work together to promote angiogenesis (the formation of new blood vessels). In low oxygen (hypoxic) conditions, HIF-1α becomes stable and binds to the VEGF gene, increasing VEGF production. VEGF then stimulates the growth of new blood vessels. HIF-1α is also increased by oncogenes (cancer-causing genes) and growth factors through the ERK signaling pathway. This leads to the activation of enzymes that break down the basement membrane, which supports cells. As a result, cell adhesion molecules (like cadherin and vimentin) are lost, and cells become more mobile. Additionally, hypoxia triggers the release of growth factors like TGF and c-MET, promoting cell growth and proliferation. These processes highlight HIF-1α's key role in tumor growth and progression. Solid tumours are hypoxic, so what? Low oxygen levels in solid tumors (hypoxia) are linked to several issues: Increased Metastasis and Poor Tumor Grade: Hypoxia often leads to more aggressive cancer and worse outcomes.  Mutations in p53: Hypoxia puts pressure on tumors to develop mutations in the p53 gene, which is crucial for controlling cell growth and repair. Increased Mutations: Hypoxic conditions can cause more genetic mutations. Gene Expression Changes: Hypoxia stimulates the expression of genes that help cancer cells grow and survive, including vascular endothelial growth factor (VEGF), which promotes new blood vessel formation. Neovascularisation in tumours Neovascularization in tumors involves the formation of new blood vessels to supply the growing tumor with oxygen and nutrients. This process, known as tumor angiogenesis, was recognized in the early 1970s. Angiogenesis: Tumor cells release signals that prompt the formation of new blood vessels from existing ones. Vessel Abnormalities: New tumor blood vessels often have structural defects, irregular blood flow, and areas of low oxygen. Tumor Growth and Spread: Tumors depend on these new blood vessels to survive, grow, and spread. Understand the vasculature, understand the tumour Understanding the blood supply of a tumor—its vasculature—is crucial for comprehending tumor behavior and growth. Angiogenesis, the formation of new blood vessels from existing ones, involves four key steps in endothelial cells: 1. Breaking Through the Basal Lamina: Endothelial cells, which line blood vessels, must penetrate the basal lamina, a layer of extracellular matrix surrounding blood vessels. 2. Migration Towards Source Signal: These cells then migrate towards signals that indicate where new vessels are needed.  3. Proliferation: The endothelial cells proliferate, or multiply, to form more cells that will make up the new blood vessels. 4. Formation of Tubes: Finally, the proliferated cells organize themselves into tube-like structures, forming new blood vessels. VEGF (Vascular Endothelial Growth Factor) is a crucial factor in this process. It plays a major role in angiogenesis and is strongly linked to tumor malignancy and metastasis. VEGF-A specifically attracts new blood vessels to areas of low oxygen (hypoxic regions) within the tumor, supporting tumor growth and spread throughout its lifetime. Chaotic solid tumour neovascularisation Chaotic solid tumor neovascularization refers to the disorganized and irregular formation of new blood vessels within a tumor. As a tumor grows, it requires more blood supply, leading to increased neovascularization. However, this new blood vessel formation is often abnormal, resulting in several issues: 1. Leaky Vasculature: The newly formed blood vessels are often structurally irregular and poorly formed, which leads to increased permeability. This means that these vessels leak more easily, allowing fluids and proteins to escape into the surrounding tissue. 2. Increased Interstitial Pressure: The leakage of fluids from the leaky vessels accumulates in the interstitial space (the area between cells), increasing the pressure within the tumor tissue. This elevated interstitial pressure can further disrupt normal blood flow and exacerbate the chaotic nature of the tumor’s blood supply. In a solid tumor, red indicates well-oxygenated arterial blood, blue represents poorly oxygenated venous blood, and green shows lymphatic vessels. This color-coding helps visualize the different types of blood and fluid circulation within the tumor. From hypoxia to tumour invasion Tumor invasion involves several critical steps, especially in the context of low-oxygen conditions: 1. Proteolytic Modification of the Extracellular Matrix (ECM): Tumor cells produce enzymes that break down the ECM, the supportive network around cells. This modification allows tumor cells to move through the surrounding tissue. 2. Migration: Tumor cells then migrate through the altered ECM to spread to other areas. 3. Loss of Cell–Cell Adhesion: Tumor cells lose their adhesion to neighboring cells, which facilitates their detachment and movement. In low-oxygen (hypoxic) conditions, Hypoxia-Inducible Factor (HIF) triggers the expression of lysyl oxidase (LOX). LOX is a protein that inhibits E-cadherin, a molecule crucial for cell adhesion. Reduced E-cadherin levels weaken cell-cell connections, promoting tumor cell invasion and spread. Chemotherapy effectiveness can be limited by the tumor microenvironment in several ways: 1. Drug Penetration: The structure and composition of the tumor microenvironment—such as the abnormal blood vessel network and high interstitial pressure—can hinder the ability of chemotherapy drugs to penetrate deep into the tumor tissue. 2. Drug Distribution: Even if drugs enter the tumor, they might not reach all tumor cells at high enough concentrations to be effective. The irregular and leaky blood vessels within tumors can lead to uneven drug distribution. These factors can result in insufficient drug exposure for some tumor cells, reducing the overall effectiveness of chemotherapy. Exploiting the tumour microenvironment: mechanisms and therapeutic strategies Tumor heterogeneity within the microenvironment significantly impacts how sensitive tumor cells are to drug treatments. This heterogeneity includes: 1. Regions of Hypoxia: Areas with low oxygen levels can reduce the effectiveness of some chemotherapy drugs, as low oxygen can limit the drug's ability to induce cell death. 2. Chaotic Blood Supply: Irregular and poorly functioning blood vessels can affect drug delivery, leading to uneven distribution of the drug within the tumor. Some areas may receive inadequate drug concentrations. 3. Acidity: Tumors often have an acidic microenvironment due to high metabolic activity. This acidity can alter the effectiveness of drugs, affecting their ability to kill cancer cells. Designing drugs that better address these factors involves creating treatments that: Penetrate the Tumor Effectively: Develop drugs that can reach deeper and more uniformly throughout the tumor. Target Hypoxic Cells: Design drugs that are more effective in low-oxygen conditions or that can be activated in hypoxic regions. Adapt to Acidic Environments: Formulate drugs that remain active or become more effective in acidic conditions. These strategies aim to overcome the limitations imposed by the tumor microenvironment and enhance the overall effectiveness of cancer treatments. Exploiting Hypoxia: Mechanisms and Therapeutic Strategies Oxygen Tension: Tumors often have areas with low oxygen levels (hypoxia), which differ significantly from normal tissues. This low oxygen tension affects how tumor cells respond to treatment. Resistance of Hypoxic Cells: Hypoxic cells in tumors are often resistant to standard therapies due to the following reasons: Free Radical Formation: Many anticancer drugs, like Doxorubicin, work by generating free radicals that damage DNA. These drugs rely on oxygen to facilitate the transfer of electrons to produce these radicals. In low oxygen conditions, this process is less efficient, leading to reduced cytotoxicity (cell-killing effect) of these drugs. Overcoming Resistance: 1. Selective Agents for Hypoxic Cells: Develop drugs that are specifically toxic to hypoxic cells, circumventing the reduced efficacy of conventional drugs in low-oxygen environments. 2. Hypoxia-Activated Prodrugs: Some drugs, like Mitomycin C and experimental agents, require reduction (activation) under hypoxic conditions to become effective. These drugs remain inactive in well-oxygenated areas but become potent in hypoxic regions of the tumor, targeting those resistant cells. By focusing on these strategies, therapies can be designed to better target and kill the hypoxic cells that often contribute to tumor resistance and treatment failure. Summary Reduced Nutrient Supply and Waste Removal lead to an acidic environment. Hypoxia (low oxygen) triggers the release of VEGF to promote new blood vessel formation (neo-angiogenesis). Invasion is facilitated by lysyl oxidase (LOX), which represses E-cadherin, weakening cell adhesion. Resistance to Therapy: For example, drugs like Doxorubicin are less effective in hypoxic conditions where oxygen-dependent mechanisms are impaired. Lecture 6 The Warburg hypothesis The Warburg hypothesis posits that cancer cells predominantly use aerobic glycolysis, rather than mitochondrial oxidative phosphorylation, for glucose metabolism. This upregulation of glycolysis results in increased glucose consumption, even in the presence of oxygen, which is less efficient for ATP production. This paradoxical preference for glycolysis is thought to be an early, irreversible step in the development of cancer, facilitating tumor growth and progression. Warburg was incorrect While Warburg's observations about cancer cell metabolism were correct, he was mistaken in attributing cancer primarily to altered metabolism. We now understand that cancer is fundamentally driven by genetic mutations that lead to uncontrolled cell growth. These genetic changes often result in metabolic alterations, such as increased glycolysis, to support rapid cell proliferation. Warburg's experiments were not flawed; rather, his conclusions about the cause of cancer were incomplete. Modern research has integrated his findings into a broader understanding of cancer metabolism, recognizing that genetic mutations and metabolic changes are both crucial aspects of cancer development. Does the tumour microenvironment select for altered metabolism? Tumor microenvironment selects for altered metabolism: The harsh conditions within the tumor microenvironment, such as low oxygen levels and limited nutrient availability, drive the selection of cells that can thrive under these stresses. This often includes cells that utilize alternative metabolic pathways, such as glycolysis, to sustain their energy needs. Tumor cells have little access to oxygen: Tumor growth often outpaces the development of blood vessels, leading to hypoxic (low oxygen) regions within the tumor. This lack of oxygen forces cells to adapt their metabolic processes to survive. Glucose diffusion advantage: Glucose can diffuse more efficiently than oxygen within the tumor. Because of this, tumor cells often rely on glycolysis, which does not require oxygen, to convert glucose into energy. This adaptation helps them maintain energy production even in hypoxic conditions. Affinity for aerobic glycolysis: The preference for aerobic glycolysis, despite the presence of oxygen, remains a key characteristic of cancer cells. This phenomenon, known as the Warburg effect, is not fully explained by hypoxia alone. It is believed that this metabolic switch supports rapid cell growth and proliferation by providing both ATP and essential biosynthetic precursors for cell division. The Warburg effect The Warburg effect: The conversion of glucose to lactic acid in the presence of oxygen, known as aerobic glycolysis, is a hallmark of cancer cells. This phenomenon, termed the Warburg effect, contrasts with normal cells, which rely on oxidative phosphorylation for energy production when oxygen is available. Warburg’s hypothesis: Otto Warburg hypothesized that the primary cause of cancer was a fundamental change in cellular metabolism, where cancer cells preferentially use glycolysis over oxidative phosphorylation, even in the presence of sufficient oxygen. He proposed this metabolic switch as an early and irreversible step leading to cancer. Explanation: The Warburg effect is partly due to tumors being poorly vascularized, resulting in low oxygen supply. To survive, cancer cells adapt by relying on glycolysis to convert glucose to lactate, providing energy through non-oxidative pathways. This adaptation allows them to thrive in hypoxic conditions and supports rapid cell growth and proliferation. Glucose metabolism in normal mammalian cells: 1. In the presence of oxygen: ○ Pyruvate, derived from glucose, undergoes oxidation in the mitochondria through the citric acid cycle and oxidative phosphorylation, producing 36 ATP per glucose molecule for a total yield of 38 ATP. 2. In the absence of oxygen: ○ Pyruvate is reduced to lactate in the cytoplasm through anaerobic glycolysis. Lactate is then exported from the cell. 3. Hydrogen ions and acidification: ○ Both aerobic and anaerobic glycolysis generate hydrogen ions, which contribute to the acidification of the extracellular space as they are exported from the cell along with lactate. Positron-emission tomography (PET) with 18fluorodeoxyglucose (18F-FdG) imaging: Quantification of glucose uptake and glycolysis: PET imaging with 18F-FdG is used to measure the rate of glucose uptake and glycolysis in tissues. FdG tracer: 18F-FdG is a glucose analogue that, once inside cells, is phosphorylated by hexokinases and becomes trapped, allowing for detection. Radiolabelling: The trapped 18F-FdG emits positrons, enabling the visualization of tissues with high glucose uptake, such as most cancers. FdG PET imaging: Both primary and metastatic cancers show significantly increased glucose uptake, making this imaging technique useful for cancer detection. Specificity and sensitivity: The ability of FdG PET to identify primary and metastatic cancer lesions is high, with near 90% specificity and sensitivity. Limitations: Sensitivity is reduced for small lesions, and specificity is affected because other tissues, like immune cells, also uptake FdG 18F-FdG PET and the re-emergence of Warburg: Correlation with outcomes: High levels of 18F-FdG uptake are strongly associated with poorer patient outcomes and elevated lactate levels. Lack of correlation with hypoxia: 18F-FdG uptake in cancers does not directly correlate with hypoxic markers, indicating that cancer cells can consume glucose at high rates even when oxygen is present. Gene overexpression: Genes involved in glycolysis and glucose uptake are significantly overexpressed in cancer cells. Renewed focus on Warburg effect: These findings have brought the Warburg effect and the phenomenon of aerobic glycolysis back into focus for many researchers, highlighting its importance in cancer metabolism. NB.The Warburg and Pasteur effects: Pasteur effect: In mammalian cells, the presence of oxygen inhibits glycolysis because mitochondria oxidize pyruvate to CO2 and H2O, efficiently producing ATP. Metabolic versatility: Cells must be able to switch between metabolic pathways to maintain energy production under varying conditions. Warburg effect: Unlike normal cells, cancer cells exhibit increased aerobic glycolysis, even in the presence of oxygen. This leads to higher glucose uptake and is associated with poorer clinical outcomes. Hallmark of cancer: The Warburg effect is now recognized as a genuine hallmark of cancer, reflecting the unique metabolic adaptations of cancer cells. mTOR – sensor of metabolic state of cell AMPK – sensor of O2 in cell The mTOR pathway: Cancer mutations: Growth and survival signals, often mutated in cancer, heavily influence the mTOR pathway. Key players: Major tumor suppressors and proto-oncogenes, such as PI(3)K, Akt, and PTEN, significantly impact mTOR activity. Activation: Nutrients and growth factors inhibit the TSC2/TSC1 complex, which then releases its inhibition on Rheb. Activated Rheb signals the mTOR/raptor complex to promote protein synthesis. Inhibition: Energy depletion (via LKB1/AMPK), hypoxia (via HIF-1/REDD), or lack of amino acids activate TSC2/TSC1. This inhibition of Rheb and mTOR decreases protein synthesis and triggers autophagy. Systematic analysis of protein-protein interactions (PPI) in autophagy and neurodegenerative diseases: Objective: Create detailed PPI networks connecting huntingtin (htt), its known partners, and proteins involved in autophagy. Methodology: Use high-throughput automated yeast two-hybrid (Y2H) screening to identify interactions. Human cDNAs encoding htt proteins, interaction partners, and autophagy proteins will be subcloned into Y2H vectors. Automated  interaction assays will be performed using robots. Validation: Confirm identified interactions through pull-down, co-immunoprecipitation, aggregation assays, and functional autophagy tests. Interactions will be scored for confidence. Integration: Combine high-confidence PPI networks with expression profiling and proteomics data, in collaboration with D. Rubinsztein from the University of Cambridge. Outcome: Identify critical protein complexes involved in htt misfolding and degradation in Huntington's disease. Data will be stored in a collaborative database for ongoing use. Oncogenic driver’s of the Warburg effect Role of oncogenes: Oncogene activation or tumor microenvironment changes can drive the Warburg effect, where cancer cells rely on aerobic glycolysis even in the presence of oxygen. Adaptive advantages: These alterations provide cancer cells with significant benefits, such as enhanced adaptability, increased proliferation, and improved survival, contributing to tumor growth and resilience. Oncogenic drivers of cancer metabolism: PI3K signaling pathway: ○ Enhances glucose uptake: Promotes increased glucose uptake by mobilizing glucose transporters to the cell surface. ○ Activates HK2: Hexokinase 2 is activated to phosphorylate and trap glucose inside the cell. Ras and Myc transcription factors: ○ Increase glucose consumption: Up-regulate metabolic genes, boosting glycolysis and glucose uptake.  ○ Direct activation: Bind to and activate genes encoding glycolytic enzymes, including HK2. Hypoxia-inducible factors (HIF): ○ Decrease oxygen dependence: Activate target genes that reduce reliance on oxygen, supporting glycolysis in hypoxic conditions. Loss/mutation of p53: ○ Uncoupling glycolysis from oxygen: May contribute to altered metabolic regulation, similar to the Warburg effect. Drawbacks of using aerobic glycolysis: Toxic metabolite buildup: Aerobic glycolysis leads to the accumulation of lactate and other toxic metabolites, which need to be removed from the cell. Acidification: Increased lactate production contributes to the acidification of the tumor microenvironment, which can impact surrounding tissues and immune response. High energetic demand: Cancer cells have a high energy requirement and often rely on diverse fuel sources, such as glutamine, to meet their metabolic needs beyond what glycolysis alone can provide. So there is increased glucose uptake, but why? Proliferative and survival advantages: High glucose uptake supports the biosynthesis of macromolecules, such as nucleic acids and lipids, which are essential for rapid cell growth and division. Prevention of cell death: Increased glucose uptake helps reduce reactive oxygen species (ROS) levels, which can otherwise lead to apoptosis (cell death). By managing ROS levels, cancer cells enhance their survival and resistance to stress. Bioenergetics in cancer: ATP production: Recent studies show that cancers derive about 17% of their ATP from glycolysis, similar to normal tissues (~20%), and often rely more on oxidative phosphorylation for energy. Energy sources: Cancer cells primarily use oxidative metabolism of glutamine and glucose (both anaerobic and aerobic glycolysis) for ATP production. Proliferation needs: The need for ATP to support oncogene-induced proliferation is recognized, but increased glycolysis alone does not fully explain the energy needs of cancer cells. Glycolytic phenotype: The preference for glycolysis in cancer cells may be driven by factors other than just ATP production, indicating additional benefits or adaptations associated with this metabolic phenotype. Acidosis and its growth advantage to tumors: Proliferative advantage: Acidic conditions in the tumor microenvironment are optimized for the tumor's growth while harming surrounding competing cells. Facilitates invasion: Acidosis promotes tumor invasion by stimulating the degradation of the extracellular matrix and enhancing angiogenesis (formation of new blood vessels), aiding in tumor spread and growth Lactate: Aerobic and hypoxic symbiosis: HIF-1 role: Under hypoxic conditions, HIF-1 induces proteins that boost glucose uptake (GLUT1), enhance glycolysis (glycolytic enzymes), produce lactate and H+, and facilitate their removal from the cell (via CA9, NHE1, MCT4). Efficient glucose use: By shuttling lactate to aerobic cells, tumors make the most of the limited glucose available. Lactate functions: Lactate serves as a signal for tumor invasion and metastasis, and helps stabilize HIF-1α, further supporting the tumor's adaptation. Inhibition effect: Targeting MCT1 with inhibitors in tumor-bearing mice can be effective in killing cancer cells by disrupting lactate transport. Lactate and endothelial cells: Export and uptake: Lactate is exported from tumor cells by MCT4 and taken up by endothelial cells through MCT1. ROS production: Once inside endothelial cells, lactate stimulates reactive oxygen species (ROS) production via NADPH oxidase and blocks the PHD2-mediated inhibition of IKK. IKK activation: Activated IKK leads to the phosphorylation and degradation of IκB, which releases NF-κB. NF-κB and IL-8: NF-κB upregulates IL-8, which promotes endothelial cell migration, proliferation, tube formation, and angiogenesis, facilitating tumor growth and spread. Why would cancer cells use an ineffective way to produce energy? Pyruvate kinase (PK-M2) – embryonic form Pyruvate kinase: forcing glycolytic products into the citrate acid cycle Phosphoenolpyruvate- conversion from glucose to lactate with the production of energy Pyruvate kinase (PK-M2) – embryonic form PK-M2 in normal and cancer cells: PK-M2 (tetramer): In normal cells, PK-M2 exists as a tetramer with high affinity for phosphoenolpyruvate (PEP), efficiently converting glucose to lactate while producing energy. PK-M2 (dimer): In cancer cells, PK-M2 often forms a dimer with low affinity for PEP. This causes the accumulation of phosphometabolites above pyruvate kinase, which are diverted into synthetic pathways for producing essential building blocks like nucleic acids, phospholipids, and amino acids. Warburg effect: This shift contributes to the Warburg effect, where cancer cells increase glycolysis and lactate production despite having adequate oxygen, to support rapid growth and proliferation. Summary Acidosis: Increased glucose uptake leads to higher glycolysis and lactate production, causing acidosis, which gives tumors a growth advantage by creating a more favorable microenvironment for themselves while harming surrounding cells. Glycolysis (Warburg effect): Enhanced glycolysis (Warburg effect) in tumors results in the production of lactate and the synthesis of essential building blocks like nucleotides and fatty acids. This supports rapid cell proliferation and growth. PK-M2 role: PK-M2, in its dimer form, helps divert glycolytic intermediates into pathways for synthesizing these crucial building blocks, further supporting tumor growth.