Research Project Flashcards
71
Project Title:Targeting replication stress for cancer therapy
Main project supervisor: Dr. Lisiane Meira
Other supervisor(s) (if any): PhD student Levy Gregory
Please select the main project type(s): Laboratory
Project summary: (overview of background, aims, and experimental approach; provide as much detailed information as possible so the students have a good idea of what the project involves, approximately 200-300 words)
Our genomes must be faithfully copied once, and only once, every cell cycle. It is therefore not surprising that DNA replication is a highly regulated process. Importantly, any obstacle to replication can compromise the faithful copying of the genome and induce replication stress. Several obstacles, including DNA damage, may interfere with DNA replication, causing replication stress (Gaillard et al.,2015).
In this project you will work with cancer cells that were treated with several DNA damaging agents, in addition to agents that disrupt nucleotide metabolism. Your job will be to prepare proteins from those cells, quantify the proteins and using Western blotting, you will detect replication stress and/or the activation of the DNA Damage Response.
Western blotting is a challenging technique, but we will use different antibodies and different conditions and different cell lines so we are expected to get positive detection of the required events at least for some conditions/antibodies/cell lines. And we will troubleshoot when needed.
- How are our genomes copied once every cell cycle?
The process of copying our genomes once every cell cycle is known as DNA replication. This occurs during the S phase (Synthesis phase) of the cell cycle. Here’s a simplified overview of how it works:
Initiation: DNA replication begins at specific locations in the genome called origins of replication. Proteins called origin recognition complexes (ORCs) bind to these origins to start the process1.
Unwinding: The enzyme helicase unwinds the double-stranded DNA, creating two single strands that serve as templates for replication1.
Priming: Short RNA primers are synthesised by the enzyme primase. These primers provide a starting point for DNA synthesis1.
Elongation: DNA polymerases add nucleotides to the RNA primers, synthesising new DNA strands complementary to the original templates. This occurs in a semi-conservative manner, meaning each new DNA molecule consists of one old strand and one new strand12.
Leading and Lagging Strands: DNA synthesis occurs continuously on the leading strand and discontinuously on the lagging strand, forming short segments called Okazaki fragments. These fragments are later joined together by the enzyme DNA ligase12.
Proofreading and Repair: DNA polymerases have proofreading abilities to correct errors during replication. Additional repair mechanisms ensure the fidelity of DNA replication2.
Termination: Once the entire genome is replicated, the process concludes, and the cell proceeds to the next phases of the cell cycle, eventually leading to cell division2.
This highly regulated process ensures that each daughter cell receives an accurate copy of the genome, maintaining genetic stability across generations23.
https://www.mbi.nus.edu.sg/mbinfo/genome-regulation/how-is-dna-replicated/
- What is replication stress?
Replication stress in cells refers to the challenges and obstacles that occur during the DNA replication process, which can lead to stalled replication forks and potential genome instability12. Here are some key points about replication stress:
Causes: Replication stress can be caused by various factors, including DNA damage, insufficient replication factors, conflicts between replication and transcription, and unusual DNA structures1.
Consequences: When replication forks stall, it can lead to incomplete DNA replication, which may cause mutations or chromosomal rearrangements if not properly resolved12.
Cellular Response: Cells have evolved mechanisms to detect and respond to replication stress. Proteins like ATR and ATM are activated to stabilize stalled replication forks and initiate repair processes1.
Relevance to Disease: Persistent replication stress is linked to various diseases, including cancer, as it can contribute to genomic instability23.
https://link.springer.com/article/10.1007/s00412-016-0573-x
- How can replication stress be used as a potential target for cancer therapy?
Replication stress can be exploited as a potential target for cancer therapy due to the unique vulnerabilities it creates in cancer cells. Here’s how:
Cancer Cell Vulnerability: Cancer cells often experience high levels of replication stress due to rapid and uncontrolled cell division, loss of tumor suppressors, and defects in DNA repair mechanisms12. This makes them more dependent on the replication stress response pathways to survive.
Targeting Key Proteins: Therapeutic strategies can involve targeting proteins that are crucial for managing replication stress. For example, inhibiting kinases like ATR, CHK1, WEE1, and MYT1, which coordinate the DNA damage response and cell cycle control, can selectively kill cancer cells by exacerbating their replication stress3.
Synthetic Lethality: This approach involves targeting pathways that cancer cells rely on due to their existing genetic defects. For instance, cancer cells with BRCA mutations are more sensitive to PARP inhibitors, which further impair their ability to repair DNA, leading to cell death1.
Combination Therapies: Combining replication stress-inducing agents with other treatments, such as immunotherapies, can enhance the overall therapeutic effect. This is because replication stress can modulate the cell-intrinsic immune response, making cancer cells more susceptible to immune attack1.
Enhancing Replicative Stress: Some strategies aim to further increase replication stress in cancer cells to a level that they cannot manage, leading to catastrophic failure of cell proliferation4.
By leveraging these strategies, researchers and clinicians aim to selectively target cancer cells while sparing normal cells, thereby improving the efficacy and safety of cancer treatments1
https://aacrjournals.org/cancerres/article/79/8/1730/640966/Exploiting-DNA-Replication-Stress-for-Cancer
- What is glioblastoma and why are new treatment options needed?
Glioblastoma is a highly aggressive type of brain tumor that originates from glial cells, which support and protect neurons in the brain. It is the most common and fast-growing malignant brain tumor in adults12. Here are some key points about glioblastoma and why new treatment options are crucial:
Characteristics of Glioblastoma
Aggressiveness: Glioblastomas are classified as grade IV tumors, indicating their rapid growth and tendency to infiltrate surrounding brain tissue2.
Symptoms: Symptoms can include headaches, seizures, cognitive and personality changes, and neurological deficits, depending on the tumor’s location2.
Diagnosis: Diagnosis typically involves imaging studies like MRI and biopsy to confirm the tumor type and grade2.
Current Treatment Options
Surgery: The primary treatment is surgical removal of as much of the tumor as possible. However, complete removal is often challenging due to the tumor’s invasive nature12.
Radiation Therapy: Post-surgery, radiation therapy is used to target remaining cancer cells1.
Chemotherapy: Temozolomide is the standard chemotherapy drug used alongside radiation1.
Need for New Treatment Options
Limited Efficacy: Despite aggressive treatment, glioblastoma has a poor prognosis. The median survival time is about 15 months, and only a small percentage of patients survive beyond five years12.
Tumor Recurrence: Glioblastomas often recur after initial treatment due to their highly invasive nature and resistance to conventional therapies1.
Resistance to Therapy: Glioblastoma cells can develop resistance to chemotherapy and radiation, making it difficult to achieve long-term control of the disease1.
Side Effects: Current treatments can cause significant side effects, impacting the patient’s quality of life1.
Research and New Therapies
Targeted Therapies: Research is ongoing to develop therapies that specifically target molecular pathways involved in glioblastoma growth and survival1.
Immunotherapy: This approach aims to harness the body’s immune system to fight the tumor. Clinical trials are exploring various immunotherapeutic strategies1.
Gene Therapy: Experimental treatments are being developed to correct genetic mutations associated with glioblastoma1.
Replication Stress-Targeted Therapies: As discussed earlier, targeting the replication stress response in glioblastoma cells is a promising area of research1.
New treatment options are essential to improve survival rates and quality of life for glioblastoma patients. Ongoing research and clinical trials are critical to finding more effective and less toxic therapies.
https://www.medicalnewstoday.com/articles/glioblastoma-treatment#what-treatments-are-available
and
https://www.cancerresearchuk.org/about-cancer/brain-tumours/types/glioblastoma/
- What is the potential of using replication stress targeted cancer therapies in glioblastoma and the challenges and / or side effects associated with this?
Targeting replication stress in glioblastoma (GBM) therapy holds significant potential due to the unique vulnerabilities of glioblastoma stem-like cells (GSCs). Here’s a detailed look at the potential and challenges:
Potential of Replication Stress-Targeted Therapies in GBM
Exploiting GSC Vulnerabilities: Glioblastoma stem-like cells exhibit high levels of replication stress and an upregulated DNA damage response (DDR), which contribute to their resistance to conventional therapies like radiation and temozolomide12. By targeting these stress responses, therapies can selectively kill GSCs, potentially preventing tumor recurrence.
Key Targets: Inhibitors of ATR and PARP have shown promise in preclinical studies. These inhibitors can exacerbate replication stress in GSCs, leading to cell death and overcoming radioresistance12. This approach leverages the inherent weaknesses in the cancer cells’ replication machinery.
Combination Therapies: Combining replication stress-targeted therapies with other treatments, such as radiation or chemotherapy, can enhance their effectiveness. This multi-pronged approach can help in tackling the tumor more comprehensively1.
Challenges and Side Effects
Toxicity: One of the main challenges is the potential toxicity to normal cells. Since replication stress-targeted therapies can affect rapidly dividing cells, they might also harm healthy tissues, leading to side effects like bone marrow suppression and gastrointestinal issues3.
Drug Resistance: Cancer cells can develop resistance to these therapies by activating alternative pathways or mutating target genes. This adaptive resistance can limit the long-term efficacy of the treatments3.
Biomarker Identification: Identifying reliable biomarkers to predict which patients will benefit from replication stress-targeted therapies is crucial but challenging. Without accurate biomarkers, it’s difficult to tailor treatments to individual patients3.
Complexity of Tumor Biology: The heterogeneity of glioblastoma tumors means that not all cells within a tumor will respond uniformly to replication stress-targeted therapies. This complexity requires a nuanced approach to treatment3.
Clinical Validation: Ensuring that these therapies are effective in clinical settings, beyond preclinical models, is essential. This involves extensive clinical trials to validate safety and efficacy3.
Conclusion
While replication stress-targeted therapies offer a promising avenue for treating glioblastoma, particularly by targeting the resilient GSCs, they come with significant challenges. Addressing these challenges requires ongoing research, careful patient selection, and combination strategies to maximize therapeutic benefits while minimising side effects.
https://aacrjournals.org/cancerres/article/78/24/6713/631890/Replication-Stress-An-Achilles-Heel-of-Glioma
- How can Western Blot be used to detect replication stress and/or the activation of the DNA Damage Response?
Western Blot is a powerful technique used to detect specific proteins in a sample, making it highly useful for studying replication stress and the activation of the DNA Damage Response (DDR). Here’s how it works in this context:
Detecting Replication Stress
Markers of Replication Stress:
Proteins such as phosphorylated RPA (pRPA) and γH2AX are commonly used markers. pRPA indicates single-stranded DNA regions, while γH2AX marks sites of DNA damage1.
Sample Preparation: Cells are treated with replication stress-inducing agents like hydroxyurea (HU) or aphidicolin (APH). These agents stall replication forks, leading to the accumulation of replication stress markers2.
Protein Extraction: Proteins are extracted from the treated cells and separated using gel electrophoresis based on their size1.
Blotting and Detection: The separated proteins are transferred to a membrane and probed with specific antibodies against pRPA and γH2AX. The presence and intensity of these bands indicate the level of replication stress1.
Detecting DNA Damage Response Activation
DDR Proteins: Key proteins involved in the DDR, such as ATR, CHK1, and their phosphorylated forms (pATR, pCHK1), are targeted. These proteins are activated in response to replication stress and DNA damage1.
Western Blot Procedure: Similar to detecting replication stress, cells are treated to induce DNA damage, proteins are extracted, separated, and transferred to a membrane1.
Antibody Probing: The membrane is probed with antibodies specific to DDR proteins and their phosphorylated forms. The detection of these proteins indicates activation of the DDR1.
Example Application
In a study, researchers treated cells with hydroxyurea (HU) to induce replication stress and then used Western Blot to detect increased levels of γH2AX and pRPA, confirming the induction of replication stress and activation of the DDR32.
Challenges and Considerations
Specificity: Ensuring the antibodies used are specific to the target proteins is crucial for accurate detection.
Quantification: The intensity of the bands on the Western Blot can be quantified to measure the extent of replication stress and DDR activation.
Controls: Including appropriate controls is essential to validate the results.
Western Blot remains a valuable tool for studying the molecular mechanisms underlying replication stress and the DNA Damage Response, providing insights into cellular responses to genomic instability.
https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-019-5934-4
READING
(Gaillard et al.,2015).
Genome instability is a hallmark of cancer, and DNA replication is the most vulnerable cellular process that can lead to it. Any condition leading to high levels of DNA damage will result in replication stress, which is a source of genome instability and a feature of pre-cancerous and cancerous cells. Therefore, understanding the molecular basis of replication stress is crucial to the understanding of tumorigenesis. Although a negative aspect of replication stress is its prominent role in tumorigenesis, a positive aspect is that it provides a potential target for cancer therapy.
The goal of shedding light on the mechanisms underlying the initiation of an oncogenic process, should open up new possibilities for cancer diagnostics and treatment.
https://pubmed.ncbi.nlm.nih.gov/25907220/ OR
https://www.nature.com/articles/nrc3916
Genome instability and cell cycle dysregulation are commonly associated with cancer. DNA replication stress driven by oncogene activation during tumorigenesis is now well established as a source of genome instability. Replication stress generates DNA damage not only during S phase, but also in the subsequent mitosis, where it impacts adversely on chromosome segregation. Some regions of the genome seem particularly sensitive to replication stress-induced instability; most notably, chromosome fragile sites. In this article, we review some of the important issues that have emerged in recent years concerning DNA replication stress and fragile site expression, as well as how chromosome instability is minimized by a family of ring-shaped protein complexes known as SMC proteins. Understanding how replication stress impacts on S phase and mitosis in cancer should provide opportunities for the development of novel and tumour-specific treatments.
https://www.sciencedirect.com/science/article/pii/S1044579X17302778?via%3Dihub
Genomic instability is considered as one of the hallmarks of cancer. In normal cells, various checkpoints could either activate DNA repair or induce cell death/senescence. Cancer cells on the other hand potentiate DNA replicative stress, due to defective DNA damage repair mechanism and unchecked growth signaling. Though replicative stress can lead to mutagenesis and tumorigenesis, it can be harnessed paradoxically for cancer treatment. Herein, we review the mechanism and rationale to exploit replication stress for cancer therapy. We discuss both established and new approaches targeting DNA replication stress including chemotherapy, radiation, and small molecule inhibitors targeting pathways including ATR, Chk1, PARP, WEE1, MELK, NAE, TLK etc. Finally, we review combination treatments, biomarkers, and we suggest potential novel methods to target DNA replication stress to treat cancer.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564951/
DNA replication is one of the fundamental biological processes in which dysregulation can cause genome instability. This instability is one of the hallmarks of cancer and confers genetic diversity during tumorigenesis. Numerous experimental and clinical studies have indicated that most tumors have experienced and overcome the stresses caused by the perturbation of DNA replication, which is also referred to as DNA replication stress (DRS). When we consider therapeutic approaches for tumors, it is important to exploit the differences in DRS between tumor and normal cells. In this review, we introduce the current understanding of DRS in tumors and discuss the underlying mechanism of cancer therapy from the aspect of DRS.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5797825/
Replication stress is a feature of precancerous (6) and cancerous cells (7). Cancer cells exhibit heightened replication stress response, for example through CHEK1 amplification, to support rapid proliferation and tolerate the higher levels of replication stress (8). Replication stress itself and the mechanisms that mitigate replication stress are increasingly recognized as cancer cell–specific vulnerabilities that could be exploited therapeutically (9–12). However, rational targeting of these dependencies requires reliable approaches to assess replication stress and its cellular response in patient tumors. Measures of replication stress—including ssDNA or ssDNA-bound RPA levels, phosphorylated form of histone H2AX (γH2AX)—are widely used in experimental settings (13, 14), but are not optimized for use in large cohorts of clinical tumor samples.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9648410/
Papers from Lisi
- The novel phosphatase NUDT5 is a critical regulator of triple-negative breast cancer growth:
https://breast-cancer-research.biomedcentral.com/articles/10.1186/s13058-024-01778-w
Summary:
Background: TNBC is a highly aggressive form of breast cancer that lacks the estrogen receptor (ER), progesterone receptor (PR), and HER2, making it difficult to treat with targeted therapies.
Objective: The study aimed to explore NUDT5 as a potential therapeutic target for TNBC.
Methods: Researchers used various techniques, including mRNA expression analysis, siRNA targeting, and small molecule inhibition, to study the effects of NUDT5 on TNBC cells. They also used animal models to assess tumor growth.
Findings: The study found that NUDT5 is overexpressed in TNBC and plays a significant role in regulating oxidative DNA damage. Inhibiting or knocking down NUDT5 suppressed TNBC cell growth by increasing oxidative DNA lesions and triggering a DNA damage response, which ultimately inhibited cell proliferation.
Conclusion: NUDT5 is crucial for TNBC cell proliferation and survival. Targeting NUDT5 could be a promising therapeutic strategy for treating TNBC1.
- Causes and consequences of replication stress
https://www.nature.com/articles/ncb2897
Summary:
Replication Stress Definition: Replication stress is a complex phenomenon that affects genome stability and cell survival. It involves the formation of aberrant replication fork structures containing single-stranded DNA.
Role of ATR: The kinase ATR (ATM- and Rad3-related) is crucial in the replication stress response. It stabilises and helps restart stalled replication forks, preventing DNA damage and genome instability.
Sources of Replication Stress: The article highlights various sources of replication stress, both intracellular and extracellular, that can disrupt DNA replication.
Cellular Response Mechanisms: Cells have developed pathways to manage replication stress, including the activation of dormant replication origins to ensure the completion of DNA replication.
Implications for Human Disease: Understanding replication stress responses is important for diagnosing and treating diseases caused by defective responses to replication stress.
Genome Duplication: The DNA replication machinery must balance accuracy, speed, and resource distribution to complete replication efficiently. Eukaryotic cells regulate the firing of replication origins to manage this process.
Evolving Definition: The definition of replication stress is continually evolving due to its complex nature and the variety of sources and repercussions involved.
- Targeted NUDT5 inhibitors block hormone
signalling in breast cancer cells
https://www.nature.com/articles/s41467-017-02293-7
Summary:
NUDT5 Function: NUDT5 is involved in ADP-ribose metabolism and plays a crucial role in hormone-dependent gene regulation and proliferation of breast cancer cells1.
Inhibitors Identified: The researchers identified potent inhibitors of NUDT5, with the lead compound being TH54271.
Mechanism of Action: TH5427 blocks progestin-dependent nuclear ATP synthesis, which is essential for chromatin remodelling, gene regulation, and cell proliferation1.
Implications: The study suggests that targeting NUDT5 with inhibitors like TH5427 could be a promising strategy for treating hormone-dependent breast cancer1.
- Hallmarks of DNA replication stress
https://www.sciencedirect.com/science/article/pii/S1097276522004385?via%3Dihub
Summary:
Definition and Causes: DNA replication stress refers to the slowing or stalling of replication forks, which can be caused by various factors such as DNA damage, difficult-to-replicate regions, and oncogene activation1.
Consequences: Replication stress can lead to chromosomal instability (CIN), which includes both structural (s-CIN) and numerical (n-CIN) changes in chromosomes. This instability is a key driver of cancer and other diseases1.
Mechanisms: The article highlights mechanisms like mitotic rescue of replication stress (MRRS) and centriole disengagement, which can either prevent or contribute to chromosomal aberrations and segregation errors1.
Outcomes: Errors in chromosome segregation can result in micro nucleation and aneuploidy, promoting inflammation, senescence, or chromothripsis. These outcomes can reduce cellular fitness in non-cancerous cells but fuel genomic instability in cancer cells1.
Therapeutic Implications: Understanding the mechanisms of replication stress and CIN can help in developing targeted cancer therapies that exploit these vulnerabilities1
Undergraduate Research Project - Relevant Info and Overview
We will be researching into Glioblastoma > one of the most common and aggressive brain cancers in adults. We will be working with the U87 human glioblastoma cell line which is a MODEL for glioblastoma. The cells are derived from a 44 year old male patient.
The U87 cells show a spindle like morphology and they are adherent - meaning they grow anchored to a solid support > the U87 cells are grown attached to some sort of plastic in culture.
Why are we researching into Glioblastoma?
