230316 Flashcards
What is the difference between genetic and epigenetic changes in a cancer? Give a sample to
describe how they work together in cancer development?
Genetic Changes:
Definition: Genetic changes refer to alterations in the DNA sequence itself. These can include mutations, deletions, duplications, insertions, and chromosomal rearrangements.
How they work in cancer: Genetic changes can lead to the activation of oncogenes (e.g., KRAS mutations) or the inactivation of tumor suppressor genes (e.g., TP53 mutations). These mutations can lead to uncontrolled cell growth, resistance to apoptosis, and other hallmarks of cancer.
Example: Mutations in the KRAS gene, which encodes a signaling protein involved in cell proliferation, can cause constant activation of the protein, leading to continuous cell division and cancer development (e.g., pancreatic cancer).
Epigenetic Changes:
Definition: Epigenetic changes are modifications to the DNA or associated proteins that do not change the DNA sequence but affect gene expression. These modifications include DNA methylation, histone modification (acetylation, methylation), and chromatin remodeling.
How they work in cancer: Epigenetic changes can silence tumor suppressor genes or activate oncogenes without altering the underlying DNA sequence. For example, hypermethylation of tumor suppressor genes like CDKN2A (which encodes p16) can silence its expression, promoting tumorigenesis.
Example: In breast cancer, the promoter region of the BRCA1 gene is often hypermethylated, leading to its silencing and increasing the risk of tumorigenesis, even though there might not be any genetic mutation in the BRCA1 gene itself.
How Genetic and Epigenetic Changes Work Together in Cancer Development
In many cancers, genetic and epigenetic changes work together to promote tumorigenesis, with genetic mutations driving the basic transformation of cells and epigenetic modifications further fine-tuning the gene expression landscape to support cancer progression.
Example: Colorectal Cancer:
Genetic Change: A mutation in the APC gene (adenomatous polyposis coli), a tumor suppressor, is an early event in the development of colorectal cancer. This mutation leads to the activation of the Wnt signaling pathway, driving uncontrolled cell proliferation.
Epigenetic Change: In addition to the genetic mutation in APC, epigenetic alterations, such as the hypermethylation of the MLH1 promoter, occur in later stages of colorectal cancer. This silences the MLH1 gene, which is involved in mismatch repair. The silencing of MLH1 results in a failure to repair DNA replication errors, contributing to the accumulation of further mutations (a phenomenon called microsatellite instability, MSI).
Combined Effect: The APC mutation initiates tumorigenesis by activating cell growth, while the MLH1 epigenetic silencing accelerates tumor progression by allowing additional genetic mutations to accumulate. Together, these genetic and epigenetic changes lead to the development of colorectal cancer.
What do hallmarks of cancers mean? List two hallmarks and further describe their important roles
in cancer development.
Two Key Hallmarks of Cancer:
Sustaining Proliferative Signaling:
Description: Cancer cells can continuously signal themselves or surrounding cells to promote their own growth and division, bypassing the normal regulatory mechanisms that control cell proliferation. This hallmark involves the activation of oncogenes, which are genes that normally promote cell division but are mutated or overexpressed in cancer to result in uncontrolled cell growth.
Key Mechanisms: Cancer cells can activate growth factor receptors (e.g., EGFR, HER2) or aberrantly activate downstream signaling pathways like RAS/RAF/MAPK or PI3K/AKT to sustain proliferative signals. Mutations or overexpression of signaling molecules (e.g., KRAS mutation) can lead to constitutive signaling even in the absence of external growth factors.
Importance in Cancer Development: This hallmark is fundamental for tumor growth because it allows cancer cells to avoid the normal growth-regulatory signals that would typically limit cell division. As a result, tumors can grow uncontrollably and expand over time, contributing to cancer progression.
Evading Growth Suppressors:
Description: Cancer cells acquire the ability to evade or bypass the normal growth inhibitory signals that regulate cell cycle progression and suppress tumorigenesis. Tumor suppressor genes, such as TP53 (which encodes p53) or RB1 (which encodes the retinoblastoma protein), are commonly mutated or inactivated in cancer cells, allowing uncontrolled cell division.
Key Mechanisms: Cancer cells can inactivate tumor suppressors via mutations, deletions, or epigenetic silencing. For instance, p53, a critical tumor suppressor involved in the DNA damage response, may be mutated or inactivated, preventing it from inducing cell cycle arrest, senescence, or apoptosis in response to genetic damage. Similarly, the RB1 gene, which controls the G1-to-S phase checkpoint of the cell cycle, may be lost, allowing the cell to bypass normal growth control.
Importance in Cancer Development: The loss of growth suppressors allows cancer cells to bypass critical checkpoints in the cell cycle and evade apoptosis (programmed cell death). This enables tumor cells to accumulate mutations, proliferate uncontrollably, and avoid the regulatory mechanisms that would normally prevent cancer development.
Explain the heterogeneity of a cancer, and give two methods which can detect the heterogeneity
of cancer cells.
Tumor heterogenity is observed both between the primary tumor and the metastasis in the patient but also within the tumor itselfs. Since cancer cells are highly mutational different cells within the tumor can have aquired different mutations, epigenetic changes, etc.
The enviorment for the tumor is also different in different parts, some cancer cells will be closer to blood cells while other will experience more hypoxia, others will be more accesible for the immune system while others will be less.
There are also different cell types within the tumor there are cancer stem cells that self renew and differentiate etc.
There is also a surrvival bias from treatments so those cells who are more resistant towards the treatment can surrvive and continue to proliferate.
The enviorment are also different between the tumors some will live in highly metabolic areas, aeorobic etc while other’s will live in aneorobic sites so they will have to develop differently to surrvive in their own enviorment.
Describe typical morphological features of an apoptotic cell. Name two proteins which are
involved in apoptosis and briefly describe their signaling pathways in cancer development.
Typical Morphological Features of an Apoptotic Cell:
Apoptosis, also known as programmed cell death, is a highly regulated and controlled process that enables the removal of damaged or unnecessary cells without causing an inflammatory response. The morphological features of an apoptotic cell are distinct and include:
Cell Shrinkage: The cell loses its volume and becomes smaller due to the loss of water and ion homeostasis.
Chromatin Condensation: The chromatin becomes highly condensed and aggregates at the nuclear periphery, which is visible under a microscope.
Nuclear Fragmentation: The nucleus undergoes fragmentation, resulting in the breakdown of nuclear material into smaller pieces.
Membrane Blebbing: The cell membrane forms small protrusions or “blebs” as the cytoskeleton collapses, causing the cell shape to become irregular.
Formation of Apoptotic Bodies: The cell breaks up into several small, membrane-bound fragments known as apoptotic bodies. These bodies are then phagocytosed by surrounding cells or macrophages, preventing inflammation.
Loss of Membrane Integrity: Apoptotic cells exhibit loss of phospholipid asymmetry, and the outer leaflet of the plasma membrane becomes enriched with phosphatidylserine, which acts as a “eat me” signal for phagocytes.
These morphological changes are regulated by the signaling pathways that control apoptosis and can be observed using various techniques such as flow cytometry (Annexin V staining), microscopy, and DNA fragmentation assays.
Proteins Involved in Apoptosis and Their Signaling Pathways in Cancer Development:
p53 (Tumor Suppressor Protein):
Normal Function: p53 is a critical tumor suppressor that responds to cellular stress (e.g., DNA damage, oncogene activation) by inducing cell cycle arrest, DNA repair, senescence, or apoptosis. When DNA damage is irreparable, p53 initiates apoptosis to eliminate damaged cells, preventing the development of cancer.
In Cancer: In many cancers, p53 is mutated or inactivated (e.g., TP53 mutations), allowing cells with damaged DNA to evade apoptosis. This loss of p53 function contributes to genomic instability and tumor progression. Without the apoptosis-inducing activity of p53, cells that should be eliminated can continue to proliferate and accumulate further mutations, driving cancer development.
Signaling Pathway: p53 activates apoptosis through the upregulation of pro-apoptotic proteins like BAX and PUMA, which promote mitochondrial outer membrane permeabilization (MOMP) and the release of cytochrome c, leading to caspase activation.
Bcl-2 Family Proteins:
Normal Function: The Bcl-2 family includes both pro-apoptotic (e.g., BAX, BAK) and anti-apoptotic (e.g., Bcl-2, Bcl-xL) proteins that regulate mitochondrial integrity and the intrinsic apoptotic pathway. The balance between pro- and anti-apoptotic members determines whether a cell undergoes apoptosis in response to stress signals.
In Cancer: In many cancers, anti-apoptotic Bcl-2 family proteins are overexpressed, which prevents the activation of the intrinsic apoptotic pathway, allowing cancer cells to survive under conditions that would otherwise induce cell death. This overexpression contributes to resistance to chemotherapy and radiation therapy.
Signaling Pathway: In response to stress signals, pro-apoptotic proteins like BAX and BAK oligomerize and translocate to the mitochondria, where they induce MOMP and the release of cytochrome c. Cytochrome c then activates caspase-9, which in turn activates caspase-3 to execute apoptosis. Anti-apoptotic proteins like Bcl-2 block this process by preventing the activation of BAX and BAK.
The greatest mortality in cancer is often the metastasis, that is, from primary tumour to a
secondary site. Briefly describe the process of EMT (epithelial–mesenchymal-transition) that
occurs during this process.
How to identify cancer stem cells? Why is it important to identify cancer stem cells in
chemotherapy for cancer patients?
Epithelial–mesenchymal transition (EMT) is a crucial biological process that plays a significant role in cancer metastasis. During EMT, epithelial cells, which are normally tightly connected and exhibit polarized structures, undergo a series of molecular and morphological changes that convert them into mesenchymal-like cells. These mesenchymal cells are more migratory, invasive, and capable of surviving in the bloodstream, enabling them to spread from the primary tumor to secondary sites in the body.
Key Features of EMT in Cancer Metastasis:
Loss of Epithelial Characteristics:
Cell-Cell Adhesion: Epithelial cells express E-cadherin, a cell adhesion molecule that maintains tight cell–cell junctions. During EMT, the expression of E-cadherin is downregulated, and the cells lose their tight junctions, which reduces their attachment to one another.
Loss of Polarity: Epithelial cells exhibit apical–basal polarity. EMT leads to a loss of this polarity, which is essential for the cells to adopt a more mesenchymal phenotype.
Tight Junction Disruption: Downregulation of tight junction proteins (e.g., claudins) during EMT allows the cells to detach from the surrounding extracellular matrix (ECM) and from other epithelial cells.
Gain of Mesenchymal Features:
Increased Motility: The cells gain the ability to move through the ECM by expressing mesenchymal markers such as vimentin, fibronectin, and N-cadherin, which help in cell migration and invasion. This change makes the cancer cells more adaptable and capable of invading tissues.
Degradation of ECM: Mesenchymal-like cells can secrete proteases, such as matrix metalloproteinases (MMPs), which degrade the ECM, allowing the cells to invade surrounding tissues and move through tissue barriers to reach blood vessels.
Increased Resistance to Apoptosis:
Survival Pathways: EMT involves the activation of survival pathways, such as those regulated by TGF-β, Wnt/β-catenin, and Notch signaling. These pathways help the mesenchymal-like cells survive in the circulation and avoid apoptosis, which is critical for their metastatic spread.
Induction by Tumor Microenvironment:
EMT can be induced by signals from the tumor microenvironment, including factors such as transforming growth factor-beta (TGF-β), epidermal growth factor (EGF), and hypoxia. These factors activate transcription factors, such as Snail, Slug, Twist, and ZEB1/2, that drive the EMT process.
The activation of these transcription factors results in the repression of epithelial markers and the induction of mesenchymal markers.
Process of EMT in Metastasis:
Invasion of Local Tissue: Cancer cells undergoing EMT lose their adhesive properties, allowing them to dissociate from the primary tumor and invade the surrounding tissue. They also gain the ability to degrade the ECM, which facilitates movement through tissue barriers.
Intravasation: Mesenchymal-like tumor cells can invade nearby blood vessels or lymphatics (intravasation), entering the bloodstream or lymphatic system and traveling to distant sites.
Survival in Circulation: Once in circulation, the cells face challenges such as immune surveillance and physical stress. However, cells undergoing EMT often exhibit increased survival characteristics, allowing them to persist in the bloodstream and resist apoptosis.
Extravasation and Colonization: After reaching a distant organ, the mesenchymal-like cells can exit the blood vessels (extravasation) and colonize secondary sites, forming metastases. These cells may revert to an epithelial phenotype at the secondary site, a process known as mesenchymal–epithelial transition (MET), where they re-establish cell–cell junctions and start proliferating to form metastatic tumors.
Describe the function of telomeres and telomerases in cancer development.
Telomerase activation which is an enzyme that catalyzes the extension of telomers and has a subunit that serves as a template for the RNA component for the telomerase repeats. In a lot of cancers this is upregulated often through overexpression of the TERT gene which promoter often gets mutated to overexpress in tumors. This allows the cells to dividide indefiently.
Alternative lenghening of telomers (ALT) is telomerase independent and relies on homologous recombination to maintain telomere lenght. This involves exchange of telomeric sequences between sister chromatids or other chromosomes and incorperation of telomeric DNA synthesized using the DNA template from another telomere. Can also be upregulated in cancer but less common but leads to heterogenous telomers.
How does Wnt signalling pathway work in cancer development? Pick up at least two genes/proteins from the
pathway to describe their roles in cancer development.
In the canonical pathway, the binding of Wnt ligands (such as Wnt1 or Wnt3a) to Frizzled receptors activates intracellular signaling cascades that lead to the stabilization and accumulation of β-catenin. This accumulation allows β-catenin to enter the nucleus, where it interacts with T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors, ultimately activating the expression of genes involved in cell proliferation, survival, and differentiation. In normal cells, this pathway is tightly regulated to prevent uncontrolled growth, but in cancer cells, aberrant activation of Wnt signaling contributes to oncogenesis.
Genes/Proteins Involved in Wnt Signaling and Their Roles in Cancer Development
β-catenin (CTNNB1):
Normal Function: β-catenin is a key downstream effector of the Wnt signaling pathway. In the absence of Wnt signaling, β-catenin is phosphorylated by the destruction complex (including proteins like APC and GSK-3β), leading to its degradation in the proteasome. When Wnt ligands bind to the Frizzled receptors, β-catenin stabilizes and accumulates in the cytoplasm, then translocates to the nucleus where it acts as a co-activator for transcription factors like TCF/LEF to promote gene expression.
Role in Cancer: In many cancers, β-catenin is mutated or stabilized inappropriately, leading to its accumulation and activation of target genes even in the absence of Wnt signaling. This results in uncontrolled cell proliferation and survival, contributing to tumorigenesis. Mutations in CTNNB1, which encode β-catenin, are frequently found in various cancers such as colorectal cancer, liver cancer, and melanoma. These mutations often lead to the loss of phosphorylation sites, preventing β-catenin from being degraded, thereby keeping the pathway continuously activated.
Example: In colorectal cancer, mutations in the APC gene (a negative regulator of β-catenin) lead to the accumulation of β-catenin in the cell, which promotes the expression of oncogenes such as c-Myc and cyclin D1, driving cell proliferation.
APC (Adenomatous Polyposis Coli):
Normal Function: APC is a tumor suppressor that plays a central role in the regulation of the Wnt/β-catenin pathway. It forms part of the destruction complex that constantly degrades β-catenin under normal conditions. APC binds to β-catenin and recruits other components (such as GSK-3β and Axin) to facilitate its phosphorylation and subsequent proteasomal degradation.
Role in Cancer: Mutations in APC lead to the loss of its regulatory function, resulting in the accumulation of β-catenin and activation of Wnt target genes that promote cell division and survival. APC mutations are a key event in the development of familial adenomatous polyposis (FAP), an inherited condition that predisposes individuals to colorectal cancer. In sporadic colorectal cancers, APC mutations also contribute to the early stages of tumorigenesis.
Example: FAP is characterized by the development of numerous benign polyps in the colon due to mutations in the APC gene. These polyps are at high risk of progressing to malignant adenocarcinomas, largely because of the persistent activation of β-catenin signaling.
Implications of Wnt Signaling in Cancer
Uncontrolled Cell Proliferation: Dysregulation of the Wnt/β-catenin pathway, especially through mutations in key components like APC or β-catenin, leads to the unchecked activation of genes that promote cell cycle progression and survival. This drives continuous cell division, contributing to tumor formation.
Metastasis and Invasion: Wnt signaling also plays a role in epithelial-mesenchymal transition (EMT), a process that allows cancer cells to acquire motility and invasiveness, facilitating metastasis. By upregulating EMT-related genes (such as Snail and Twist), Wnt signaling can promote the ability of cancer cells to invade surrounding tissues and spread to distant organs.
Stemness and Cancer Stem Cells: Wnt signaling is important for maintaining the stem-like properties of certain cancer cells, known as cancer stem cells (CSCs). These cells are thought to be responsible for tumor initiation, progression, and resistance to therapy. By promoting the self-renewal and differentiation potential of CSCs, Wnt signaling contributes to the persistence and recurrence of cancer.
Name two drugs (antibodies) of immunotherapy to explain their mechanisms for attacking cancer
cells.
- Pembrolizumab (Keytruda) – PD-1 Inhibitor
Mechanism:
Target: Pembrolizumab targets the Programmed Cell Death Protein 1 (PD-1) receptor on T cells.
Normal Function of PD-1: PD-1 is an immune checkpoint receptor expressed on T cells, which, when bound by its ligands (PD-L1 and PD-L2), suppresses T cell activity. This mechanism is used by tumors to evade immune surveillance by essentially turning off T cell responses.
Action in Cancer: Pembrolizumab blocks the interaction between PD-1 and its ligands, preventing the “brake” on the immune system and allowing T cells to remain active and capable of attacking tumor cells.
Clinical Use: Pembrolizumab is used to treat various cancers, including non-small cell lung cancer (NSCLC), melanoma, and head and neck squamous cell carcinoma, by enhancing the ability of the immune system to recognize and kill cancer cells.
2. Rituximab (Rituxan) – CD20 Monoclonal Antibody
Mechanism:
Target: Rituximab targets CD20, a cell surface protein expressed on B cells, including malignant B cells in certain types of lymphoma and leukemia.
Normal Function of CD20: CD20 is involved in the regulation of B cell activation and growth. It is present on normal B cells, as well as on B cells involved in lymphoma and leukemia.
Action in Cancer: Rituximab binds to CD20 on the surface of B cells and induces several immune responses, including:
Antibody-Dependent Cellular Cytotoxicity (ADCC): The binding of Rituximab recruits immune cells such as natural killer (NK) cells to kill the targeted cancerous B cells.
Complement-Dependent Cytotoxicity (CDC): Rituximab activates the complement system, leading to the lysis of the targeted cancer cells.
Direct apoptosis: Rituximab can also induce apoptosis (programmed cell death) in B cells by binding directly to CD20.
Clinical Use: Rituximab is commonly used in the treatment of non-Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), and some autoimmune diseases by targeting malignant B cells.
Suppose that you could re-design your Lab practice of the tumour biology course with the same
aim under an unlimited condition (regardless of time consuming, material cost etc.), how could
you change (add) your setting of DNA sequencing, Western blotting and flow cytometry, and
why?
