Control of aggressive tumor cells

Each cell contains two different “tools” to repair single or double DNA breaks, which can be caused by factors such as environmental toxins, chemotherapy, or ionizing radiation. The first is made up of the DNA repair genes BRCA1 and BRCA2, while the second is an enzyme called poly (ADP-ribose) polymerase, or PARP1 for short. These tools are used by both healthy and malignant cells. They eventually do the same thing and can replace each other. If DNA repair fails because the damage is too extensive, the cell begins its own suicide program – programmed cell death – and destroys itself. This process has become a target of cancer treatments.

People who carry BRCA1 and/or BRCA2 gene mutations have a very high risk of developing certain types of tumors, most notably breast, ovarian and prostate cancer. These individuals lack one of these tools, which explains why they are more likely to develop cancerous cells. These cancer cells also have only one tool at their disposal – PARP1 – to ensure their survival. However, BRCA1/2-associated tumors are usually very aggressive and difficult to treat. So researchers led by Professor Klaus Schederet of the Max Delbrück Center, the latest author of the study, took a closer look at the signaling pathway that activates the PARP1 enzyme. In the process, they figured out a way to target this tool and render it useless.

To prevent apoptosis after DNA damage, a key signaling pathway called NF-κB is initiated. Gene transcription begins in the cell nucleus that eventually activates PARP1 so that the cell can repair the damage. By turning off the genes one by one, Ahmet Tofan, co-first author of the study along with Katina Lazarov of Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), scanned the entire human genome for genes that regulate this signaling pathway. To do this, it first inserts a ‘measure gene’ into the genome, which provides a blueprint for a fluorescent protein tag. “A few hours after treatment with an etoposide chemotherapeutic agent that caused DNA damage, the cells light up green because NF-κB is activated,” Tovan explains. Too many 20,000 gene samples lit up — but some didn’t. This was due to turning off a gene that plays an important role in the signaling pathway in these samples.

Hindering PARP1 repairs

But which one? The number of candidate genes was in the thousands—after all, the NF-κB signaling pathway controls a wide range of cell functions, such as the immune response. “We used bioinformatics to eliminate those who perform routine ‘housekeeping’ tasks in the cell, and then subjected the remaining 500 genes to the same procedure again.” The team again used etoposide to induce DNA damage. “For each gene, we ran parallel tests with the cytokine TNF-alpha, which also activates this signaling pathway, but only plays a role when there is inflammation,” Schedrett says.

After that, only a small set of genes remained. The researchers already knew that some of them were part of the signaling pathway, but not others. Which one was critical? Tovan used algorithms to search various databases for clues. Where are the products of these genes mentioned besides others? Were there any research papers claiming that the proteins in question join with other proteins to form complexes? The team also combed through the lab’s databases. Then, suddenly, they found what they were looking for: Tumor Susceptibility Gene 101 (TSG101). This gene was not a new discovery in itself; It plays many well-known roles in the cell. But in this particular process, it binds to PARP1 once the enzyme has docked at the site of damage. Only then can PARP1 itself become active.

“The PARP1 is like a loaded pistol,” Schedrett says. “Whether you just scratch the cells, squeeze them with shear pressure, or break them completely, PARP1 is activated. But if there’s no TSG101 in the cell, it won’t work. It’s missing the trigger, so to speak.” The researchers were able to watch what was happening live under a microscope using fluorescent stained PARP1. A few seconds after they had burned tiny holes in the cell nucleus with a laser, the cells lit up green as PARP1 spilled into the cell nucleus on all sides and attached to sites of DNA damage. After a few minutes, the glare subsided.

amazing note

“PARP1 modifies itself, attracts other helper proteins that do the necessary repairs, and then dissociates itself,” Schedritt explains. However, if the TSG101 gene was turned off, PARP1 flushed into the sites of DNA damage in the cell nucleus at the same speed—but the green glow remained. PARP1 was not able to be deregulated from damaged DNA. “Of all the observations I made during my live-cell imaging experiments, this was the most surprising,” Tofan says. “In TSG101-null cells, PARP1 becomes trapped at sites of DNA damage.”

“Through this research, we have shown that administration of PARP inhibitors and turning off TSG101 have the same effect,” Schedritt summarizes. Using different breast cancer cell lines, the researchers were also able to demonstrate that cells without the TSG101 gene died quickly after chemotherapy. However, the fastest cells that perished were those with BRCA1 mutations, as they no longer had any DNA repair tools.

PARP inhibitors have already been used for several years in the treatment of some cancers – for example, in treatments for breast cancer patients who have a proven BRCA mutation. “Unfortunately, no single inhibitor has been developed that specifically targets PARP1, as there is a full set of PARP genes,” Schedrett says. “However, based on our results, a targeted search can now begin for therapeutic agents that block TSG101’s binding to PARP1.” Thus, the study may pave the way for the future development of highly effective and more targeted therapies for BRCA1/2-related cancers.

More information

Schedrett Laboratory, Signal Transduction in Cancer Cells

literature

Ahmed Bogra Tofan et al. (2022): “TSG101 binds to PARP1 and is essential for DNA damage-induced granulation and activation of NF-κB,” EMBO . magazineDOI: 10.15252 / embj.2021110372

Downloads

3D culture of human breast cancer cells, with DNA stained in blue and protein in the cell surface membrane stained in green.

This image was originally submitted as part of the 2015 NCI Cancer Close Up project and was selected for display. See also visualsonline.cancer.gov/closeup.

© NCI Center for Cancer Research, National Cancer Institute, National Institutes of Health, CC BY-NC 2.0.0 Update

Contacts

Professor Klaus Schedrett

Max Delbrück Center for Molecular Medicine at the Helmholtz Society (MDC)

+49- (0) 30-9406-3816

scheidereit@mdc-berlin.de

Christina Anders

Communications editor

Max Delbrück Center for Molecular Medicine at the Helmholtz Society (MDC)

+49- (0) 30-9406-2118

christina.anders@mdc-berlin.de or presse@mdc-berlin.de

Max Delbrück Center

The Max Delbrück Center for Molecular Medicine of the Helmholtz Society (Max Delbrück Center) is one of the world’s leading biomedical research institutions. Max Delbrück, a native of Berlin, was a Nobel laureate and one of the founders of molecular biology. At sites in Berlin-Buch and Mitte, researchers from nearly 70 countries have analyzed the human system – studying the biological foundations of life from its building blocks to systems-level mechanisms. By understanding what regulates or disrupts the dynamic homeostasis of a cell, organ or the entire body, we can prevent diseases, diagnose them early, and halt their progression with customized treatments. Patients should benefit as soon as possible from basic research discoveries. Therefore, the Max Delbrück Center supports episodic creation and participates in collaborative networks. It works in close partnership with Charité – Universitätsmedizin Berlin at its jointly run Center for Experimental and Clinical Research (ECRC), the Berlin Institute of Health (BIH) at Charité, and the German Center for Cardiovascular Research (DZHK). Founded in 1992, the Max Delbrück Center employs 1,600 people and is funded by 90% of the German federal government and 10% by the state of Berlin.

www.mdc-berlin.de