Question

Mutations in tumor-suppressor genes are associated with many types of cancers. In addition, epigenetic changes (such as DNA methylation) of tumor-suppressor genes are also associated with tumorigenesis (Otani et al., 2013. Expert Rev Mol Diagn $13:445-455$ ). (a) How might hypermethylation of the $p53$ gene promoter influence tumorigenesis? (b) Knowing that tumors release free DNA into certain surrounding body fluids through necrosis and apoptosis (Kloten et al., 2013. Breast Cancer Res. $15(1):\mathrm{R}4$ ), outline an experimental protocol for using human blood as a biomarker for cancer and as a method for monitoring the progression of cancer in an individual.

Short answer

**Answer:** Hypermethylation of the p53 gene promoter can contribute to cancer development by suppressing the production of the p53 protein, which is essential for preventing tumor formation. Human blood can be used as a biomarker for cancer and monitoring cancer progression by analyzing cell-free DNA (cfDNA) for the presence of tumor-specific markers and monitoring their levels over time during treatment.

Step-by-step solution

(a) Understanding the role of hypermethylation in tumorigenesis

Hypermethylation occurs when there is an increased amount of methylation within a gene promoter region. This can lead to the silencing of tumor-suppressor genes, such as p53. When the p53 gene promoter is hypermethylated, the gene is less likely to be transcribed and translated, leading to a reduction in the production of the p53 protein. The p53 protein plays a crucial role in preventing tumor formation by repairing damaged DNA and triggering cell death when repair is not possible. Therefore, hypermethylation of the p53 gene promoter could contribute to tumorigenesis by suppressing the production of the p53 protein, thus reducing its function as a tumor suppressor.

(b) Experimental protocol for using human blood as a biomarker for cancer and monitoring cancer progression

Step 1: Sample collection Collect blood samples from patients with cancer and healthy individuals (control group) at appropriate time points. This can be done using standard phlebotomy techniques. Step 2: Isolate cell-free DNA (cfDNA) Isolate cfDNA from the blood samples using appropriate methods, such as column-based or magnetic bead-based extraction kits specifically designed for cfDNA isolation. Step 3: Quantify cfDNA Measure the concentration of cfDNA in each sample using fluorometric methods or quantitative polymerase chain reaction (qPCR). Step 4: Analyze for specific tumor markers in cfDNA Perform polymerase chain reaction (PCR) or next-generation sequencing (NGS) to analyze the presence of tumor-specific markers, such as specific mutations or methylation patterns, in the cfDNA samples. Step 5: Compare and analyze results Compare the levels of cfDNA and tumor-specific markers between cancer patients and the control group. Higher levels of cfDNA and altered tumor-specific markers in cancer patients could indicate the presence and progression of cancer. Step 6: Monitor cancer progression Repeat the analysis at different time points during the course of cancer treatment to monitor the effectiveness of the treatment and disease progression. Decreased levels of cfDNA and normalized tumor-specific markers could suggest a positive response to treatment. Step 7: Analyze results and draw conclusions Analyze the data to determine whether blood samples can serve as a reliable biomarker for cancer detection and monitoring and draw conclusions based on the results.

Key concepts

DNA methylation

DNA methylation is a process involving the addition of methyl groups to the DNA molecule, typically at cytosine bases. This alteration can modify the activity of a DNA segment without changing its sequence. When methylation occurs in the promoter region of genes like tumor-suppressor genes, it can prevent these genes from being transcribed. This is significant because tumor-suppressor genes, such as the p53 gene, play an important role in controlling cell growth and apoptosis (programmed cell death). Without proper transcription, these genes cannot produce proteins necessary to protect the cell from becoming cancerous. Therefore, increased methylation or hypermethylation in crucial areas can lead to the silencing of these protective genes, potentially leading to cancer development.

Epigenetic changes

Epigenetic changes refer to heritable alterations in gene expression that occur without a change in the DNA sequence. These changes can be influenced by a variety of factors including environment, lifestyle, and even stochastic events. DNA methylation is one form of epigenetic change. Such changes can significantly impact the behavior and fate of cells. In the context of cancer, epigenetic alterations can result in the activation or silencing of key genes, influencing tumor development, growth, and progression. Unlike genetic mutations, epigenetic changes are potentially reversible, making them a significant target for therapeutic interventions.

Biomarkers for cancer

Biomarkers are biological molecules found in blood, other body fluids, or tissues that signify a particular disease state. In cancer, biomarkers can be used for screening, diagnosis, or monitoring the progression of the disease. The presence of cell-free DNA (cfDNA) in blood, which can be derived from tumor cells, is a promising biomarker for detecting cancer and tracking its evolution. By analyzing specific features of cfDNA, such as unique mutations or methylation patterns associated with cancer cells, clinicians can gain valuable insights into a patient’s cancer status and treatment response.

p53 gene

The p53 gene is a crucial tumor-suppressor gene that encodes the p53 protein, often called the "guardian of the genome." This protein is vital for maintaining genomic stability. It helps repair damaged DNA and can induce cell death if the damage is irreparable, thus preventing the propagation of potentially cancerous cells. However, if the p53 gene's promoter region becomes hypermethylated, the gene is silenced and cannot produce the necessary protein. Consequently, cells lose a major mechanism for genomic protection, resulting in increased vulnerability to cancerous transformations.

Cancer progression monitoring

Cancer progression monitoring involves tracking changes in a patient's cancer over time, often using biomarkers such as cfDNA. By regularly assessing levels of cfDNA and concomitant changes in tumor-specific markers, healthcare providers can evaluate how well a patient's cancer is responding to treatment. Monitoring these dynamics helps in adjusting treatment plans as needed. For example, if less cfDNA and normalized markers are observed over time, it may indicate an effective treatment. Continuous monitoring is crucial for personalizing cancer therapy, ensuring that any progress or regression is promptly addressed.