230530 Flashcards
Name two proto-oncogenes and describe these in detail (how is the activity regulated, what are the
typical mechanisms of activation and what it the role in tumourigenesis and/or cell cycle regulation
if applicable)
KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog)
Normal Function:
KRAS encodes a small GTPase involved in regulating cellular processes such as growth, differentiation, and survival.
In its normal, inactive state, KRAS binds GDP. Upon stimulation by receptor tyrosine kinases (RTKs), KRAS exchanges GDP for GTP, becoming active and triggering downstream signaling pathways (such as the MAPK/ERK and PI3K/AKT pathways) that promote cell proliferation and survival.
Altered Function in Cancer:
Mutations in KRAS (e.g., in codons 12, 13, or 61) cause it to be constitutively active, meaning it remains bound to GTP even without external signals.
This leads to continuous activation of growth-promoting pathways, resulting in uncontrolled cell proliferation and survival, contributing to the development of cancers, particularly in lung, pancreatic, and colorectal cancers.
MYC (MYC Proto-Oncogene, BHLH Transcription Factor)
Normal Function:
MYC encodes a transcription factor that regulates the expression of genes involved in cell cycle progression, metabolism, and growth.
In normal cells, MYC expression is tightly controlled and transient, contributing to proper cell growth and division. MYC promotes the transcription of genes necessary for cell cycle progression, such as cyclins and CDKs.
Altered Function in Cancer:
MYC is often overexpressed or amplified in various cancers, leading to uncontrolled cell growth.
The overexpression of MYC results in the activation of genes that drive cell cycle progression even in the absence of proper growth signals, and promotes cell survival by inhibiting apoptosis. This contributes to tumor formation and metastasis.
MYC overexpression is commonly observed in breast, colon, lung, and leukemias.
What are differences between apoptosis and necrosis?
Apoptosis:
Programmed cell death.
Involves cell shrinkage, chromatin condensation, and DNA fragmentation.
Plasma membrane remains intact, preventing inflammation.
Mediated by caspases.
Necrosis:
Uncontrolled cell death due to injury or stress.
Involves cell swelling, membrane rupture, and release of cellular contents.
Triggers an inflammatory response.
Not mediated by caspases.
How do you understand the heterogeneity of a cancer (definition of tumor heterogeneity)? How
does cancer heterogeneity arise?
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.
Name at least three assays for determining apoptotic cells. And describe their mechanisms, i.e.,
why/how do the assays identify apoptotic cells?
Annexin V/Propidium Iodide (PI) Staining
Purpose: Distinguishes between live, early apoptotic, late apoptotic, and necrotic cells.
Principle:
Annexin V binds to phosphatidylserine, which is translocated from the inner to the outer leaflet of the plasma membrane during early apoptosis.
PI penetrates cells with compromised membranes, identifying necrotic or late apoptotic cells.
Caspase Activity Assays
Purpose: Measures activation of caspases, key enzymes in apoptosis.
Principle: Caspases (e.g., caspase-3, -8, -9) are activated during apoptosis, cleaving specific substrates.
TUNEL Assay (Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling)
Purpose: Detects DNA fragmentation, a hallmark of late-stage apoptosis.
Principle: Terminal deoxynucleotidyl transferase (TdT) adds labeled dUTP to DNA strand breaks.
The most common reason to mortality in cancer is metastasis of the primary tumour to a secondary
site disturbing organ function. Describe at least three main routes of metastasis.
Lymphatic Spread: Cancer cells enter lymphatic vessels and travel to regional lymph nodes, eventually reaching distant organs.
Hematogenous Spread: Cancer cells invade blood vessels and circulate through the bloodstream to distant sites.
Transcoelomic Spread: Cancer cells spread across body cavities, such as the peritoneal cavity, leading to metastasis in organs like the liver and lungs.
What functional properties do cancer stem cells have? Name and describe two functional assays
for detection of cancer stem cells.
Cancer stem cells have stem cell like proprties such as self renewal and ability to differentiate. They can intiate tumor growth by self renewing, cause tumor heterogenity with their differination, they are often more resistant to therapy due to enhanced DNA repair, drug efflux etc, they can spread to distant sites facilitating metastasis, and can even after therapy remian dormant to then reactivate cuasing cancer relapse.
Sphere Formation Assay (In Vitro Functional Assay)
Description:
CSCs have the unique ability to survive and grow under non-adherent, serum-free culture conditions that inhibit the growth of non-stem cancer cells. The sphere formation assay leverages this property to assess the presence and functionality of CSCs.
Procedure:
Plate single-cell suspensions of tumor cells in a low-adherence culture plate with a defined serum-free medium supplemented with growth factors such as EGF (epidermal growth factor) and bFGF (basic fibroblast growth factor).
Incubate the cells for several days and monitor the formation of free-floating, spherical clusters called tumor spheres.
Quantify the number and size of spheres formed, as they reflect the self-renewal and proliferative capacity of CSCs.
Applications:
Identifies and enriches CSC populations.
Evaluates the impact of drugs or genetic manipulation on CSC self-renewal.
Limitations:
Sphere formation is not exclusive to CSCs; non-CSCs can sometimes form spheres.
Culture conditions may not fully recapitulate the tumor microenvironment.
Xenograft Assay (In Vivo Functional Assay)
Description:
This approach evaluates the tumorigenic potential of CSCs by transplanting them into immunocompromised mice and assessing their ability to initiate and sustain tumor growth.
Procedure:
Isolate CSCs and non-CSCs from a tumor sample using markers associated with CSCs (e.g., CD133, CD44, ALDH). This is often done via flow cytometry or magnetic-activated cell sorting (MACS).
Inject the isolated cells into immunodeficient mice (e.g., NOD/SCID or NSG mice) subcutaneously or orthotopically into the tissue of origin.
Monitor tumor formation, growth rate, and histological characteristics over time.
Compare the tumorigenic capacity of CSCs versus non-CSCs.
Applications:
Confirms the tumorigenic potential of CSCs.
Models the role of CSCs in tumor initiation and progression.
Evaluates CSC-targeting therapies in a physiologically relevant environment.
Limitations:
Requires specialized facilities and ethical approval.
Immunocompromised mice do not fully mimic human immune-tumor interactions.
Name and describe three different mechanisms by which the tumour cells avoid getting eliminated
by the immune system.
- Immune Checkpoint Upregulation
Mechanism:
Tumor cells can exploit immune checkpoint pathways to inhibit immune responses. Immune checkpoints are regulatory pathways that normally prevent excessive immune activation and autoimmunity by inhibiting T cell function.
Cancer cells upregulate checkpoint proteins like PD-L1 (Programmed Death-Ligand 1), which binds to PD-1 (Programmed Cell Death Protein 1) on T cells. This interaction inhibits T cell activation and promotes immune tolerance to the tumor.
By expressing these checkpoints, tumor cells can effectively “turn off” the immune response that would normally target and eliminate them.
Example:
Melanoma cells frequently express PD-L1, helping them evade immune detection. - Tumor Antigen Loss or Masking
Mechanism:
Tumor cells can alter or “hide” the tumor-associated antigens (TAAs) that would normally be recognized by the immune system.
This can occur through antigen loss or downregulation, where tumor cells reduce or eliminate the expression of the surface proteins that are normally recognized by immune cells such as cytotoxic T lymphocytes (CTLs).
Alternatively, tumor cells can mask their antigens by expressing molecules like MHC class I (Major Histocompatibility Complex I) that inhibit immune cell recognition. This prevents tumor-specific T cells from detecting the tumor cells.
Example:
In some ovarian cancer cases, tumor cells lose or downregulate the expression of MHC class I molecules to avoid immune recognition. - Secretion of Immunosuppressive Factors
Mechanism:
Tumor cells often create an immunosuppressive microenvironment by secreting factors that inhibit the activity of immune cells.
TGF-β (Transforming Growth Factor Beta), IL-10 (Interleukin 10), and VEGF (Vascular Endothelial Growth Factor) are among the immunosuppressive cytokines that can be secreted by tumor cells. These factors promote the recruitment of immunosuppressive cells, such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), which further dampen the immune response against the tumor.
Additionally, TGF-β can directly inhibit the cytotoxic activity of T cells and natural killer (NK) cells.
Example:
Pancreatic cancer cells secrete high levels of TGF-β, contributing to the immunosuppressive environment and hindering immune attack.
Why doesn’t the immune system fight cancer? Give at least two examples with mechanism
explanations.
- Immune Tolerance and Exhaustion
Mechanism:
One way cancer cells avoid immune attack is by exploiting immune checkpoints that regulate immune responses and prevent autoimmune reactions. These checkpoint pathways are designed to maintain immune tolerance to self-antigens and prevent overactivation of the immune system.
Tumor cells can upregulate checkpoint molecules like PD-L1 (Programmed Death-Ligand 1) on their surface, which bind to the PD-1 receptor on T cells. This interaction inhibits T cell activation, reducing their ability to attack the tumor.
Chronic exposure to tumor antigens can also lead to T cell exhaustion, where T cells become less effective over time due to persistent stimulation by the tumor. Exhausted T cells exhibit reduced cytokine production, impaired cytotoxic activity, and reduced proliferation, rendering them ineffective in fighting the tumor.
Example:
In non-small cell lung cancer, the upregulation of PD-L1 by tumor cells leads to immune checkpoint inhibition and T cell exhaustion, allowing the tumor to escape immune surveillance. - Immunosuppressive Tumor Microenvironment
Mechanism:
Tumors often create an immunosuppressive microenvironment by secreting various cytokines and growth factors that suppress immune responses. These factors can attract and activate immune cells that inhibit anti-tumor immune responses.
For example, regulatory T cells (Tregs) are recruited to the tumor site where they suppress the activity of effector T cells and natural killer (NK) cells. Additionally, myeloid-derived suppressor cells (MDSCs) can accumulate in the tumor, further suppressing immune function.
Tumors also secrete cytokines like TGF-β (Transforming Growth Factor Beta) and IL-10, which directly inhibit the activity of cytotoxic immune cells such as T cells and NK cells.
Example:
In pancreatic cancer, the secretion of immunosuppressive factors like TGF-β creates an environment that limits the effectiveness of immune responses and promotes tumor progression.
Name three chemotherapeutic drugs used in clinics today and explain shortly the mechanism of
action of these drug.
DNA Alkylating Agents
Mechanism:
DNA alkylating agents add alkyl groups to DNA, specifically to the purine bases (guanine), leading to the formation of covalent bonds. This results in DNA strand cross-linking, preventing DNA replication and transcription.
The resulting DNA damage triggers cell cycle arrest and apoptosis, particularly in rapidly dividing cancer cells.
Example: Cyclophosphamide
Cyclophosphamide is a commonly used alkylating agent in the treatment of various cancers, including breast cancer, lymphoma, and leukemia.
Antimetabolites
Mechanism:
Antimetabolites mimic naturally occurring nucleotides or other metabolites involved in DNA and RNA synthesis.
They interfere with DNA and RNA synthesis by incorporating into the growing DNA or RNA strand, leading to faulty genetic material, or by inhibiting key enzymes required for nucleotide biosynthesis.
This results in cell cycle disruption, particularly during the S-phase, and eventually cell death.
Example: Methotrexate
Methotrexate inhibits the enzyme dihydrofolate reductase (DHFR), which is essential for the synthesis of thymidine and purines, leading to the depletion of DNA precursors and arrest of DNA synthesis. It is used in the treatment of various cancers like leukemia, breast cancer, and osteosarcoma.
Topoisomerase Inhibitors
Mechanism:
Topoisomerases are enzymes that manage the topological states of DNA during replication and transcription by inducing temporary DNA strand breaks.
Topoisomerase inhibitors prevent the re-ligation of these breaks, leading to DNA damage that cannot be repaired, resulting in apoptosis.
Topoisomerase I inhibitors cause single-strand breaks, and topoisomerase II inhibitors cause double-strand breaks.
Example: Doxorubicin (Topoisomerase II inhibitor)
Doxorubicin is used in the treatment of cancers such as breast cancer, lymphoma, and sarcoma. It inhibits topoisomerase II, causing DNA damage and preventing cancer cell division.
Microtubule Inhibitors
Mechanism:
Microtubule inhibitors interfere with the function of microtubules, which are essential components of the cytoskeleton involved in cell division (mitosis).
These drugs either prevent microtubule polymerization (stabilizers) or promote depolymerization, thereby disrupting mitotic spindle formation. This prevents the proper segregation of chromosomes during cell division, leading to cell cycle arrest in the M-phase and subsequent apoptosis.
Example: Paclitaxel
Paclitaxel stabilizes microtubules, preventing their depolymerization, which disrupts cell division. It is used in the treatment of breast, ovarian, and non-small cell lung cancer.
Assuming that you are employed at a company which is focused on new drug development against
cancer, what are your main drug targets and why?
- Oncogenes and Tumor Suppressors
Target: EGFR (Epidermal Growth Factor Receptor) and HER2
Why: Oncogenes like EGFR and HER2 are frequently overexpressed or mutated in several cancers (e.g., lung cancer, breast cancer, head and neck cancers). Targeting these receptors can inhibit tumor cell proliferation and survival. EGFR and HER2 inhibitors (e.g., erlotinib, trastuzumab) have been successful in clinical use, and further development of next-generation inhibitors can help target drug-resistant cancers.
Target: TP53 (Tumor Protein 53)
Why: p53 is a tumor suppressor protein that is mutated in a large proportion of cancers, leading to uncontrolled cell proliferation. Targeting p53 pathways or restoring p53 function in tumors could lead to apoptosis and inhibit cancer cell survival, especially in tumors with TP53 mutations. - DNA Repair Pathways
Target: PARP (Poly ADP Ribose Polymerase)
Why: PARP inhibitors are effective in cancers with BRCA1/2 mutations (e.g., ovarian, breast, and prostate cancers). By inhibiting PARP, these drugs block the repair of DNA single-strand breaks, leading to DNA damage accumulation and cell death, especially in cancer cells that are already compromised in DNA repair.
Target: Checkpoint Kinases (CHK1/CHK2)
Why: CHK1 and CHK2 are key regulators of the DNA damage checkpoint. Inhibition of these kinases can sensitize tumor cells to DNA damage, making them more susceptible to chemotherapies and radiation. Targeting checkpoint kinases in tumors with defective DNA repair pathways can improve therapeutic outcomes. - Immune Checkpoints and Immunotherapy
Target: PD-1/PD-L1 and CTLA-4
Why: Immune checkpoint inhibitors (e.g., nivolumab and pembrolizumab) have revolutionized cancer treatment by preventing immune evasion through PD-1/PD-L1 and CTLA-4 interactions. Tumors often upregulate PD-L1 to inhibit T cell activation, allowing immune evasion. By targeting PD-1/PD-L1 or CTLA-4, these drugs can restore T cell activity against cancer cells, offering potential for treating cancers like melanoma, lung cancer, and renal cancer. - Metabolic Reprogramming
Target: GLUT1 (Glucose Transporter 1)
Why: Cancer cells often rely on aerobic glycolysis (the Warburg effect) for energy production. GLUT1, a key glucose transporter, is overexpressed in many cancers. Inhibiting GLUT1 could disrupt the metabolic pathway that cancer cells depend on, starving them of the glucose necessary for survival and proliferation.
Target: IDH1/IDH2 (Isocitrate Dehydrogenase)
Why: Mutations in IDH1 and IDH2 are common in cancers such as gliomas and acute myeloid leukemia (AML). These mutations lead to the production of the oncometabolite 2-hydroxyglutarate, which drives oncogenesis. Inhibiting mutant IDH enzymes can help restore normal cellular metabolism and reduce tumor progression. - Angiogenesis
Target: VEGF (Vascular Endothelial Growth Factor) and VEGFR (Vascular Endothelial Growth Factor Receptor)
Why: Tumors often induce the formation of new blood vessels (angiogenesis) to support their growth. VEGF and its receptors (VEGFR-1, VEGFR-2) play a critical role in this process. Inhibiting VEGF or VEGFR (e.g., bevacizumab) can disrupt blood supply to the tumor, slowing down its growth and enhancing the effectiveness of other treatments like chemotherapy and radiation.
