There is evidence that MRPL13 might be one of the potential mitochondrial ribosomal proteins
(MRP) candidates that are involved in breast tumorigenesis, but its role in breast cancer has
rarely been reported.
A. What type of preliminary experimental evidence would support your initial hypothesis
that the gene MRPL13 is involved in breast cancer progression? Provide at least three
examples of experimental evidence and briefly describe the methodological procedure.
- Gene Expression Analysis in Breast Cancer Tissues and Cell Lines
Procedure:
Collect breast cancer tissue samples and adjacent normal tissues, as well as a panel of breast cancer cell lines and normal mammary epithelial cell lines.
Perform quantitative PCR (qPCR) or RNA sequencing to assess MRPL13 mRNA expression levels.
Validate with Western blotting or immunohistochemistry (IHC) to examine MRPL13 protein levels.
Correlate MRPL13 expression levels with clinical parameters (e.g., tumor grade, stage, metastasis).
Expected Evidence:
Higher MRPL13 expression in breast cancer tissues and cell lines compared to normal controls, with an association between elevated MRPL13 levels and aggressive tumor characteristics. - Functional Studies Using MRPL13 Overexpression and Knockdown
Procedure:
Use plasmids or viral vectors to overexpress MRPL13 in low-MRPL13 breast cancer cell lines.
Use siRNA/shRNA or CRISPR-Cas9 to knock down or delete MRPL13 in high-MRPL13 breast cancer cell lines.
Evaluate the impact on cancer-related phenotypes such as:
Proliferation: Use MTT or CellTiter-Glo assays.
Migration and invasion: Perform wound healing assays and Transwell migration/invasion assays.
Apoptosis: Assess with Annexin V/PI staining via flow cytometry.
Mitochondrial function: Measure mitochondrial respiration and ATP production using Seahorse XF Analyzer.
Expected Evidence:
MRPL13 knockdown should reduce proliferation, migration, and invasion, while inducing apoptosis and impairing mitochondrial function.
Conversely, MRPL13 overexpression should enhance tumorigenic traits. - In Vivo Validation Using Xenograft Models
Procedure:
Inject breast cancer cells with altered MRPL13 expression (overexpression or knockdown) into immunocompromised mice (e.g., NOD/SCID mice) either subcutaneously or orthotopically into the mammary fat pad.
Monitor tumor growth using caliper measurements and bioluminescent imaging (if using luciferase-labeled cells).
Analyze metastatic potential by assessing lung, liver, or bone metastases via histological examination or qPCR for human-specific markers.
Expected Evidence:
Tumors formed by MRPL13-overexpressing cells should grow faster, exhibit enhanced angiogenesis, and metastasize more extensively compared to controls.
MRPL13 knockdown should reduce tumor growth and metastatic burden. - Correlation With Patient Outcomes (Optional Supporting Evidence)
Procedure:
Analyze publicly available breast cancer datasets (e.g., TCGA, METABRIC) to investigate correlations between MRPL13 expression and patient outcomes, such as survival, recurrence, or response to therapy.
Expected Evidence:
High MRPL13 expression correlates with worse survival and increased recurrence rates, supporting its role in breast cancer progression.
There is evidence that MRPL13 might be one of the potential mitochondrial ribosomal proteins
(MRP) candidates that are involved in breast tumorigenesis, but its role in breast cancer has
rarely been reported.
B. Additionally, the MRPL13 was overexpressed in the MCF-7 breast cancer cell line and
the cell cycle studied. The cell cycle results as well as quantification of the cell cycle
results are shown below (Figure 1 A&B). Please answer the associated questions:
i. What should be the X- and Y-axis label?
ii. What were the cells labeled with to conduct the analysis shown in Figure 1?
iii. Which of the regions (1, 2, 3) indicate cells with 4N amount of DNA?
iv. Describe the effect of overexpressing MRPL13 on the cell cycle.
i. What should be the X- and Y-axis label?
X-axis: DNA Content
Y-axis: Number of Cells
ii. What were the cells labeled with to conduct the analysis shown in Figure 1?
The cells were labeled with a DNA-binding dye such as Propidium Iodide (PI) or DAPI, allowing DNA content quantification by flow cytometry. (?)
iii. Which of the regions (1, 2, 3) indicate cells with 4N amount of DNA?
Region 3 indicates cells with 4N DNA content, corresponding to cells in the G2/M phase of the cell cycle. (?)
iv. Describe the effect of overexpressing MRPL13 on the cell cycle.
Overexpressing MRPL13 in MCF-7 cells results in a decrease in the percentage of cells in the G1 phase (from 65% to 54%) and an increase in the S phase (from 9% to 15%) and G2/M phase (from 14% to 18%). This suggests MRPL13 overexpression promotes cell cycle progression, potentially leading to increased cell proliferation.
Figure 2 presents a model of carcinogenesis. Describe the presented model of carcinogenesis
and use it to illustrate the difference between the hereditary and sporadic form of cancer.
The Knudson’s Two-Hit Model: states that a person born with a herediatary mutation one has one healthy allele that can compensate and if they then aquire a second mutation they develop tumorigenesis while non hereditary cancers needs to develop to somatic mutations for this to develop which makes it less likely to occur.
Genomic instability is inherent to most cancers and is crucial for tumour progression. Using
PARP inhibitors as an example discuss how our understanding of molecular events that
contribute to this key cancer feature has led to novel therapeutic strategies and when the PARP
inhibitors are most effective.
Genomic instability is a hallmark of cancer, driving tumor progression by accumulating genetic mutations. PARP (Poly ADP-Ribose Polymerase) inhibitors exploit this feature by targeting DNA repair mechanisms in cancer cells1. PARP enzymes are crucial for repairing single-strand DNA breaks through the base excision repair pathway. When PARP is inhibited, single-strand breaks accumulate and eventually lead to double-strand breaks during DNA replication1.
In cells with functional homologous recombination (HR) repair mechanisms (e.g., those with intact BRCA1 or BRCA2 genes), these double-strand breaks can be accurately repaired. However, in cancer cells with defective HR repair (e.g., BRCA1 or BRCA2 mutations), the accumulation of double-strand breaks leads to cell death1. This concept is known as “synthetic lethality,” where the combination of PARP inhibition and HR deficiency results in selective killing of cancer cells while sparing normal cells.
PARP inhibitors are most effective in cancers with existing defects in HR repair, such as BRCA1/2-mutated breast and ovarian cancers. By understanding the molecular events that contribute to genomic instability and DNA repair deficiencies in cancer cells, researchers have developed PARP inhibitors as a targeted therapeutic strategy to exploit these vulnerabilities1.
Figure 3 presents a chemical modification of a cytosine. Describe the mechanism of this
process, subcellular occurrence as well as role of this process in cancer diagnostics.
DNA methylation adds a methyl group to the cytosince base of the DNA. DNMT catalyze this process and when promoter regions of tumor supressor genes are methylated they are silences. Regular genes that are activated by mutations or epigenetic changes such as losing their methylation at the promoter regions can become proto-oncogens and promote cancer.
As a diagnostic tool we can perform sequencing to find proto-oncogenes such as methylation of the BRCA1 gene, liquid biopsy to check methylation markers, and for theraputic targeting so for exampling finding supressed tumor supressor genes or prot-oncogenes and adjusting treatments based on their sensitivity.
Explain the difference between the direct action and indirect action of ionizing radiation. What
are the two main types of radiation therapy used in cancer treatment?
Direct Action: Ionizing radiation directly damages cellular DNA by breaking chemical bonds within the DNA molecule. This can cause single-strand or double-strand breaks, leading to cell death if not properly repaired.
Indirect Action: Ionizing radiation interacts with water molecules within the cell to produce reactive oxygen species (ROS), such as hydroxyl radicals. These ROS can damage cellular components, including DNA, proteins, and lipids, indirectly leading to cell death.
Types of Radiation Therapy:
External Beam Radiation Therapy (EBRT): This uses high-energy beams (like X-rays or gamma rays) directed at the tumor from outside the body.
Brachytherapy: This involves placing a radioactive source directly inside or next to the tumor, delivering a high radiation dose to the tumor while sparing surrounding healthy tissues.
Describe correlation between cancer stem cells (CSC) and circulating tumor cells (CTC). How
and where are the CTC detected in a patient? What is the therapeutic implication of CTC?
CSC and CTC Relationship:
Cancer stem cells (CSCs) are a subpopulation of tumor cells with self-renewal capabilities and the ability to differentiate into multiple tumor cell types, driving tumor initiation, progression, and metastasis.
Circulating tumor cells (CTCs) are tumor cells that detach from the primary tumor or metastatic sites and enter the bloodstream.
A subset of CTCs exhibits stem-like properties, making them highly tumorigenic and capable of forming secondary tumors at distant sites. This CSC-like behavior in CTCs contributes to metastatic spread and therapy resistance.
Key Correlation:
CSCs within the primary tumor may give rise to CTCs with enhanced metastatic potential.
CTCs that possess CSC-like traits are more likely to survive in circulation, evade immune surveillance, and establish new tumors.
How and Where Are CTCs Detected in a Patient?
Detection Methods:
Liquid Biopsy:
Blood samples are collected to isolate and analyze CTCs.
Techniques include:
Immunomagnetic enrichment: Using antibodies against tumor-specific markers (e.g., EpCAM).
Microfluidic devices: Capture CTCs based on size, deformability, or surface markers.
RT-PCR: Detect tumor-specific mRNA (e.g., CK19).
Single-cell analysis: For phenotyping and genetic profiling.
Imaging Techniques:
Advanced imaging, such as flow cytometry or fluorescence microscopy, can be used to visualize labeled CTCs.
Where CTCs Are Detected:
Bloodstream: CTCs circulate through peripheral blood, making blood draws the primary method of detection.
Bone Marrow or Lymphatic System: In some cases, disseminated tumor cells (DTCs) originating from CTCs are found in these compartments.
Therapeutic Implications of CTCs
Early Detection and Monitoring:
CTC levels can indicate the presence of metastasis before it is clinically apparent, aiding in early diagnosis.
Longitudinal monitoring of CTC levels provides information on treatment efficacy and disease progression.
Prognostic Value:
High CTC counts are associated with poor prognosis, advanced disease stage, and increased risk of metastasis.
Personalized Therapy:
Molecular profiling of CTCs allows the identification of actionable mutations or drug resistance mechanisms, guiding targeted therapy decisions.
Development of Anti-Metastatic Therapies:
Targeting CTCs with CSC-like properties may prevent metastasis by eradicating cells with high metastatic potential.
Approaches include immune-based therapies (e.g., CAR-T cells or monoclonal antibodies) or inhibitors of epithelial-to-mesenchymal transition (EMT) pathways.
List and shortly describe at least four immune parameters that can serve as biomarkers in
cancer.
Tumor-Infiltrating Lymphocytes (TILs): The presence and abundance of TILs in the tumor microenvironment can indicate the immune response against the tumor. Higher TIL levels are often associated with better prognosis and response to immunotherapy.
Programmed Death-Ligand 1 (PD-L1) Expression: PD-L1 expression on tumor cells can suppress immune responses. High PD-L1 levels are used as a biomarker for selecting patients who might benefit from immune checkpoint inhibitors (e.g., anti-PD-1/PD-L1 therapy).
Cytokine Profiles: Levels of cytokines such as interleukins (e.g., IL-6, IL-10) and interferons (e.g., IFN-γ) in the blood or tumor microenvironment can reflect the immune status and inflammation associated with cancer.
Immune Cell Subsets: The ratios of different immune cell types (e.g., CD8+ T cells, regulatory T cells) in the blood or tumor can provide information about the immune landscape and predict treatment outcomes.
A. Describe three different cellular assays used to study apoptosis in cancer
- 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.
Method:
Treat cells with the test compound.
Stain cells with fluorescently labeled Annexin V and PI.
Analyze using flow cytometry or fluorescence microscopy.
Interpretation:
Annexin V-positive, PI-negative: Early apoptosis.
Annexin V-positive, PI-positive: Late apoptosis/necrosis. - 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.
Method:
Use fluorogenic or colorimetric caspase substrates that release a detectable signal upon cleavage (e.g., DEVD-AFC for caspase-3).
Incubate cell lysates or live cells with the substrate.
Measure fluorescence or absorbance.
Interpretation: Increased caspase activity indicates apoptosis. - 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.
Method:
Fix cells or tissue samples.
Incubate with TdT and labeled dUTP.
Visualize using fluorescence microscopy or flow cytometry.
Interpretation: Cells with labeled DNA breaks are apoptotic.
Comparison and Selection
Annexin V/PI: Ideal for distinguishing apoptosis stages and assessing viability.
Caspase Activity: Useful for identifying apoptosis signaling pathways.
TUNEL Assay: Best for detecting late-stage apoptosis and DNA damage.
B. Describe briefly three non-apoptotic forms of cell death in cancer.
Necrosis: Uncontrolled cell death due to external factors (e.g., injury, toxins), leading to cell lysis and inflammation.
Autophagy: Self-digestion process where cells degrade their own components to recycle nutrients, often a survival mechanism but can lead to cell death if excessive.
Necroptosis: Programmed form of necrosis mediated by receptor-interacting protein kinases (RIPKs), leading to cell swelling and rupture.
Give example of two oncogenes and contrast the normal function of the protein encoded by the
gene as compared to the action of the form altered in cancer.
- 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.
List and briefly describe four different mechanisms of actions of the chemotherapy agents used
in cancer treatment and provide at least one example of the drug that belongs to each group.
- 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.
